Antibody based reagents that specifically recognize toxic oligomeric forms of tau

ABSTRACT

The invention relates to antibodies, antibody fragments and binding agents that specifically recognize oligomeric tau but do not bind to monomelic tau, fibrillar tau or non-disease associated forms of tau.

RELATED APPLICATION

This application claims priority under 35 U.S.C 119(e) to provisionalU.S. Ser. No. 61/713,441 filed Oct. 12, 2012, which application isincorporated hereby by reference.

BACKGROUND OF THE INVENTION

Numerous studies have implicated small soluble oligomeric aggregates ofAβ as toxic species in Alzheimer's disease (AD), and increasing evidencealso implicates oligomeric forms of tau as having a direct role indisease pathogenesis of AD and other tauopathies such as FrontotemporalDementia (FTD). As the focus of Aβ studies has slowly shifted towardsoluble Aβ species and mechanisms, new reagents were needed that couldspecifically identify the variety of different aggregate speciespresent. Indeed, many contradictory studies on the role of Aβaggregation in AD were reported and progress impeded because suitablyselective reagents were not available to characterize the aggregatespecies present. Increasing evidence from cell and animal modelsindicate that oligomeric rather than fibrillar forms of tau are toxicand correlate with neuronal degeneration, therefore well characterizedreagents that can specifically recognize the diversity of taumorphologies present in the human brain are critically needed tofacilitate studies to identify the most promising tau species for use asbiomarkers of disease and to study toxic mechanisms.

The microtubule associating protein tau is a major component of theneurofibrillary tangles associated with AD and tauopathies that arecharacterized by hyperphosphorylation and aggregation of tau. Tau playsan important role in assembly and stabilization of microtubules. Tau isa natively unfolded protein, and similar to a number of other nativelyunfolded proteins, it can aberrantly fold into various aggregatemorphologies including β-sheet rich fibrillar forms. The different typesof post-translational modifications of tau in AD includephosphorylation, glycosylation, glycation, prolyl-isomerization,cleavage or truncation, nitration, polyamination, ubiquitination,sumoylation, oxidation and aggregation. Tau has 85 putativephosphorylation sites, and excess phosphorylation can interfere withmicrotubule assembly. Tau can be modified by phosphorylation or byreactive nitrogen and oxygen species among others. Elevated total tauconcentration in CSF has been correlated with AD, as has the presence ofvarious phosphorylated tau forms, and the ratio of tau to Aβ42. Reactivenitrogen and oxygen can modify tau facilitating formation of aggregateforms including oligomeric species. Levels of oligomeric tau have alsobeen implicated as a potential early diagnostic for AD. Therefore,determination of total tau, phosphorylated tau and oligomeric tauconcentrations all have potential value as diagnostics forneurodegenerative diseases including tauopathies and AD.

Tau is an intrinsically unstructured protein due to its very lowhydrophobic content containing a projection domain, a basic proline-richregion, and an assembly domain. Hexapeptide motifs in repeat regions oftau give the protein a propensity to form β-sheet structures whichfacilitate interaction with tubulin to form microtubules as well asself-interaction to form pathological aggregates such as paired helicalfilaments (PHF). Hyperphosphorylation of tau, particularly in theassembly domain, decreases the affinity of tau to the microtubules andimpairs its ability to regulate microtubule dynamics and axonaltransport. In addition, parts of the basic proline-rich domain and thepseudo-repeat also stabilize microtubules by interacting with itsnegatively charged surface. Alternative splicing of the second, thirdand tenth exons of tau results in six tau isoforms of varying length inthe CNS. The assembly domain in the carboxyl-terminal portion of theprotein contains either three or four repeats (3R or 4R) of a conservedtubulin-binding motif depending on alternative splicing of exon 10. Tau4R isoforms have greater microtubule binding and stabilizing abilitythan the 3R isoforms. Human adult brains have similar levels of 3R and4R isoforms, whereas only 3R tau is expressed at the fetal stage. Intauopathies, mutations altering the splicing of tau transcript and theratio of 3R to 4R tau isoforms are sufficient to cause neurodegenerativedisease. Therefore tau in human brain tissue can exist in a variety ofdifferent lengths and morphologies and with multiple post-translationalmodifications.

Tau plays a critical role in the pathogenesis of AD and studies showthat reduction of tau levels in AD animal models reverses diseasephenotypes and that tau is necessary for the development of cognitivedeficits in AD models caused by over-expression of Aβ. While NFTs havebeen implicated in mediating neurodegeneration in AD and tauopathies,animal models of tauopathy have shown that memory impairment and neuronloss do not associate well with accumulation of NFT. Animal studiesshowed improvement in memory and reduction in neuron loss despite theaccumulation of NFTs, a regional dissociation of neuron loss and NFTpathology, and hippocampal synapse loss and dysfunction and microglialactivation months before the accumulation of filamentous tau inclusions.The pathological structures of tau most closely associated with ADprogression are tau oligomers. All these studies suggest that tautangles are not acutely neurotoxic, but rather that pretangle oligomerictau species are responsible for the neurodegenerative phenotype, similarto toxic role of oligomeric Aβ species.

Numerous studies suggest that extracellular tau species contribute toneurotoxicity through an “infectious” model of disease progression_(—)For example, tau pathology spreads contiguously throughout the brainfrom early to late stage disease, extracellular tau aggregates canpropagate tau misfolding from the outside to the inside of a cell, brainextract from a transgenic mouse with aggregated mutant human tautransmits tau pathology throughout the brain in mice expressing normalhuman tau, induction of pro-aggregation human tau induces formation oftau aggregates and tangles composed of both human and normal murine tau(co-aggregation), and levels of tau rise in CSF in AD, whereas Aβ levelsdecrease. A receptor-mediated mechanism for the spread of tau pathologyby extracellular tau has been described.

Collectively, these studies all indicate that aggregated oligomericspecies of tau, both intracellular and extracellular are vitallyimportant in AD and other tauopathies. In order to more clearly definethe role of individual tau forms in disease, there is a critical need todevelop a series of well-defined reagents that selectively recognizeindividual target morphologies, and to use these reagents to identifywhich tau forms are the best biomarkers for AD, which forms are involvedin toxicity both intra- and extracellularly, and which forms in braintissue and CSF samples can distinguish between healthy and AD patients.

Therefore, reagents that can specifically target tau oligomers would bevaluable tools for diagnostic and therapeutic applications for AD,frontotemporal dementia, other tauopathies and neurodegenerationfollowing traumatic brain injury.

Accordingly, there exists the need for new therapies and reagents forthe treatment of Alzheimer's disease, frontotemporal dementia, othertauopathies and neurodegeneration following traumatic brain injury, inparticular, therapies and reagents capable of effecting a therapeuticand diagnostic benefit at physiologic (e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

The present invention discloses an antibody or antibody fragment thatspecifically recognizes oligomeric tau but does not bind monomeric tau,fibrillar tau or non-disease associated forms of tau. As used herein,the phrase “specifically recognizes oligomeric tau” indicates that itdoes not bind to or recognize non-specific proteins. As used herein, theterm “antibody” includes scFv (also called a “nanobody”), humanized,fully human or chimeric antibodies, single-chain antibodies, diabodies,and antigen-binding fragments of antibodies (e.g., Fab fragments). Asused herein, the term “oligomer” refers to a dimer, trimer, tetramer,pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer ordodecamer. Accordingly, in certain embodiments, the oligomeric tau isdimeric tau, trimeric tau, tetrameric tau, pentameric tau, hexamerictau, heptameric tau, octameric tau, nonameric tau, decameric tau,undecameric tau or dodecameric tau. In certain embodiments, theoligomeric tau is dimeric tau or trimeric tau. In certain embodiments,the oligomeric tau is trimeric tau. In certain embodiments, the oligomeris soluble.

In certain embodiments, the antibody fragment does not contain theconstant domain region of an antibody.

In certain embodiments, the antibody fragment is less than 500 aminoacids in length, such as between 200-450 amino acids in length, or lessthan 400 amino acids in length.

Certain embodiments of the invention provide an antibody fragmentcomprising amino acid sequence SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. In certainembodiments, the antibody fragment comprises amino acid sequence SEQ IDNO:1, SEQ ID NO:9 or SEQ ID NO:11.

Certain embodiments of the invention provide a binding molecule thatbinds to oligomeric tau and does not bind monomeric tau, fibrillar tauor non-disease associated forms of tau, wherein the binding moleculecomprises the sequence of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19. In certainembodiments, the binding molecule comprises the sequence of SEQ ID NO:1,SEQ ID NO:9, or SEQ ID NO:11.

Certain embodiments of the invention provide an antibody or antibodyfragment as described herein, wherein said antibody fragment is isolatedaccording to a method comprising the steps of:

-   -   a. a negative panning of a scFV phage library wherein said        negative panning eliminates phage that bind to non-desired        antigens wherein said negative panning comprises serially        contacting phage with:        -   (i) a generic protein; and        -   (ii) mononeric forms of tau;    -   and monitoring the binding of said phage to the generic protein        and monomeric forms of tau using Atomic Force Microscope (AFM)        Imaging and repeating steps (i) and (ii) until no phage is        observed binding to antigen by said AFM imaging to produce an        aliquot of phage that does not bind to monomeric tau, fibrillar        tau, or non-disease associated forms of tau;    -   b. contacting the aliquot of phage that does not bind to        monomeric tau, fibrillar tau, or non-disease associated forms of        tau with tau oligomers and incubating for time sufficient to        allow binding of phage to said oligomers; and    -   c. eluting the bound phage particles from step (b).

Certain embodiments of the invention provide an antibody or antibodyfragment isolated according to a method comprising the steps of:

-   -   (a) negative panning a scFV phage library comprising serially        contacting phage with:        -   (i) a generic protein; and        -   (ii) mononeric forms of tau;

and until less than 5% of the phage is observed binding to antigen,which produces an aliquot of phage that does not bind to monomeric tau,fibrillar tau or non-disease associated forms of tau;

-   -   (b) positive panning of the aliquot from step (a) comprising        contacting the aliquot of phage from step (a) with tau        oligomers, and incubating for time sufficient to allow binding        of phage to said brain derived tau oligomers; and    -   (c) eluting the bound phage particles from step (b).

In certain embodiments, the tau oligomer used in the positive panning istrimeric tau 4N1R.

In certain embodiments, the generic protein is bovine serum albumin(BSA).

In certain embodiments, the negative panning further comprises seriallycontacting phage with brain derived control samples that do not containoligomeric tau.

In certain embodiments, the observing of the binding of the phage to theantigen is by using Atomic Force Microscope (AFM) Imaging. In certainembodiments, the negative panning is repeated until less than 0-10%phage was observed by AFM imaging as binding to antigen in step (a).

Certain embodiments of the invention provide a method of inhibiting theaggregation of tau comprising contacting a composition that comprisestau monomers with an antibody, antibody fragment or binding molecule asdescribed herein. In certain embodiments, the aggregation of tau is in acell. In certain embodiments, the aggregation of tau is in brain tissue.In certain embodiments, the contacting with an antibody, antibodyfragment or binding molecule decreases the rate of formation of tauaggregates as compared to said rate in the absence of composition orbinding molecule.

Certain embodiments of the invention provide a method of detecting thepresence of tau in a physiological sample comprising contacting a samplewith an antibody, antibody fragment or a binding molecule as describedherein and determining the binding of said composition with said tissuesample wherein binding of said composition to said tissue sample isindicative of the presence of tau oligomers in said tissue samplewherein said presence of said tau oligomers is indicative of early stageAD, frontotemporal dementia, other tauopathies or neurodegenerationfollowing traumatic brain injury. In certain embodiments, thephysiological sample is brain tissue, serum, cerebrospinal fluid (CSF),blood, urine or saliva.

Certain embodiments of the invention provide a method of preventing orinhibiting the accumulation of tau in the brain of a mammal comprisingadministering to said mammal a composition comprising an antibodyfragment or a binding molecule as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Height distribution analysis of various tau samples obtainedfrom AFM images. AD Tau #1 and AD Tau #3 represent tau samples obtainedpurified from AD brain tissue. Tau412M and Tau441M represent monomericsamples of 3R (tau 412) and 4R (tau 412) tau samples. Tau 441Orepresents and oligomeric sample of tau441. Differences in height andoligomeric state between samples are readily detected.

FIGS. 2A-2B. Lactate dehydrogenase (LDH) test of various tau species onSHSY-5Y human neuroblastoma cells. (A) Toxicity of monomeric, dimericand trimeric tau 1N4R (aka. tau 412) towards SHSY-5Y cells. (B) Toxicityof monomeric, dimeric and trimeric tau 2N4R (aka. tau 441) towardsSHSY-5Y cells. In both (A) and (B), for each group from left to right,the first bar is 3 h, the second bar is 18 h, the third bar is 24 h andthe fourth bar is 48 h.

FIGS. 3A-3C. (A) F9T scFv amino acid sequence. (B) Comparison of the DNAsequence for F9 scFv (before repair) and F9T-7 scFv (after repair). (C)DNA sequences for F9T, F9, F9T-7L and F9T-7F-RC.

FIGS. 4A-4F. DNA sequences from six scFv clones specific for trimerictau (top) and corresponding amino acid sequences (bottom), including (A)F9T; (B) D11C; (C) D4G; (D) G12C; (E) H2A; and (F) H7T.

FIGS. 5A-5B. (A) DNA sequences for C6T, F9T, D11C, D4G, G12C, H2, H2Aand H7T. (B) Comparison of the DNA sequences for C6T, F9T, D11 C, D4G,G12C, H2, H2A and H7T.

FIGS. 6A-6C. The novel biopanning process combines subtractive panningand positive panning from phagemid scFv library and the single cloningscreening using AFM. (A) Schematic panning process, in which the micacarrier can be replaced by immunotubes for bulk amount of non-desiredantigen such as BSA for rapid removal of irrelevant phage particles,especially during subtractive panning. (B) Subtractive panning againstBSA performed to eliminate non-specific phage. Left, middle and rightimages are the phage pool affinity check after BSA tube #1, #3 and #5respectively. The absence of phage binding in the right-handed sideimage denotes the accomplishment of subtractive panning against BSA. Thescale bar of 1 μm applies to all three images. (C) Positive panningagainst tau trimer was performed and imaged with AFM. A duplicate ofpositive panning compared with pure desired antigen proves that theantigen is free of phage and the phage pool depleted of non-desiredantigen binders still contains phage that specifically binds to desiredantigen. Left image is the purified trimeric tau 1N4R immobilized onmica; Right image is the same piece of mica on which the remaining phagepool of subtractive panning was deposited and non-binding particles werewashed off. The scale bar of 1 μm applies to both images.

FIG. 7. Particle size analysis of oligomeric tau captured by singleclone scFv-displayed phage from rhTau 2N4R mixed aggregates. Size ofclone phage targets compared with those of purified rhTau monomer, dimerand trimer. Individual particle capturing a phage was measured the sizeby section function in Nanoscope Analysis. The mean value of each clonephage target falls in between 2.5 nm and 3.0 nm, in accordance withrhTau 2N4R trimer size range. (Error bar:+/−standard deviation)

FIG. 8. Three clones, F9T, D11C and H2A in scFv form recognize andretain from 9-month 3×TG-AD mice hippocampus abnormal phosphorylated tauspecies that are immunoreactive with AT8. Negative control is PBS as thetarget analyte and set as 1.0 to be used as normalization standards.Signals lower than 2.0 are recorded as negative while signals above 2.0are recorded as positive. (Error bar:+/−standard deviation)

FIG. 9. The comparison of secondary antibody affinity to 9-month 3×TG-ADmice brain extracts captured by three types of primary antibody scFv.The mean comparison was performed within each group of the same primaryantibody. (Error bar:+/−standard deviation)

FIG. 10. Oligomeric tau targeted scFv clones (F9T, D11C and H2A)affinity to different 3×TG-AD mice brain extracts detected by F9TscFv-phage. Two mice for each age were tested in triplicates. The datawere grouped by the mice ages. The mean comparison was performed withineach group of the same mice age. (Error bar:+/−standard deviation)

FIG. 11A-11C. Densitometric analysis of dot blot reactivity of F9T scFvwith brain homogenates from age-matched human middle temporal gyrus(MTG). Densitometric value of dots signal is based on a scale of 0 to 1,with 0 equals the background signal and 1 equals the positive signal ofanti-pLB scFv dots. Statistical analysis is performed in one-way ANOVAcomparing means of two groups. (A) compares patients grouped byantemortem diagnosis as non-demented (ND) and Alzheimer's (AD). That ADgroup means is different from ND group mean (p<0.05) signifies F9T scFvcan detect AD from ND. (B) compares patients grouped by postmortemexamination results defined by Braak stages and neuritic plaquefrequencies directly implying the AD progression. Braak stages I-II(early stage) were both diagnosed as non-demented but half of the casesbear slight plaque compare with the other half without plaques. Braakstages III-IV (AD middle stages) display moderate plaques while Braakstages V-VI (AD late stages) display severe plaques. F9T scFv affinityto these MTG extracts directly correlates with their AD progressiondefined by Braak stages and plaque frequency. (C) Sample dot blotaffinity test of purified F9T scFv on homogenized MTG tissue fromnon-demented and Alzheimer's patients.

FIG. 12. DNA sequences of the starting region and the first heavy chainframework region (HCFR1) of selected scFvs from Sheets' library andstandard scFvs from the generic library from which Sheets' wasdeveloped. F9, H7, D4, D11 and G12 are five scFvs selected targetingrhTau 1N4R trimer, the rest are standard scFvs. Except for one missingbase pair for each clone causing frame shift, all these scFvs fromSheets' library contain the similar FR regions as those from the genericlibrary. All of these missing base pairs (highlighted in darkbackground) lie either at the beginning of HCFR1 or the connection ofHCFR1 and the methionine start codon unaffecting the restriction siteNcoI, scFv expression initiation or any complementarity-determiningregions(CDRs). Inserting the missing base pair retaining the amino acidsequences of selected clones sequences in generic library enables theseclones to express soluble scFv without interfering their epitope-bindingsites, thus maintain their specificities.

FIG. 13. Designed primers for clone sequence revision. Forward primerscontain NcoI (5′-CCATGG-3′ in italic) upstream of scFv sequence and themissing base pair(underlined). The reverse primer includes NotI(5′-GCGGCCGC-3′ in italic) downstream of scFv sequence. By performing apolymerase chain reaction using the paired primers and correspondingclone DNA template, revised clone scFv DNA fragments can be produced upto 2³⁰ copies for subcloning into E. coli and producing scFv and phage.

FIG. 14: Tau protein structural features in linear diagram. Afull-length tau protein with 441 amino acids (tau441 or tau 2N4R) isshown. Alternative splicing showed in yellow rectangles results in atotal of six isoforms, denoted by either their total number of aminoacids or the number of N′-terminal exons (Ns) and microtubule-associatedrepeats (Rs).

FIG. 15: Schematic of nonreactive monomer, reactive monomer, andreactive oligomer. Reactivity implies the ability to form anintermolecular disulfide linkage. Intramolecular disulfide linkagecauses formation of nonreactive tau monomer. The free thiols in areactive monomer allow formation of an intermolecular or intramoleculardisulfide linkage. Reactive oligomer has one or more free thiols readilyforming disulfide linkage with reactive monomeric tau for the oligomerextension purpose.

FIGS. 16A-16B: Plots of height distribution of monomeric, dimeric, andtrimeric fractions of rhTau 1N4R (a) and tau 2N4R (b).The height valueof each particle was measured using Gwyddion. The numbers of particlesfalling in continuous size ranges were calculated and normalized intocount percentages. The peak values give an approximate value for eachtau species particle size. As expected, high-degree oligomers are largerthan low-degree oligomers within the same isoform, and correspondingoligomeric aggregates from the longer isoform are larger than aggregatesfrom the shorter isoform.

FIGS. 17A-17B: Neurotoxicity of extracellular 15.5 nM monomeric,dimeric, and trimeric forms of 1N4R and 2N4R tau variants toward (a)nondifferentiated human neuroblastoma cells (SH-SY5Y) and (b)Retinoic-acid-differentiated SH-SY5Y cells was measured after 48-hourincubation using an LDH assay. For both four-repeat tau isoforms,trimeric form is more neurotoxic than monomeric and dimeric forms(P<0.001) on either neuron type. Full-length trimeric rhTau is moreneurotoxic than 1N4R trimeric rhTau. (P<0.05).

FIGS. 18A-18D: Time and concentration dependence of neurotoxicityinduced by trimeric rhTau (1N4R and 2N4R) toward neuroblastoma cellsmeasured by LDH assay. Nondifferentiated SH-SY5Y cells incubated with(a) 1N4R tau and (b) 2N4R tau; retinoic-acid-differentiated SHSY5Y cellsincubated with (c) 1N4R tau and (d) 2N4R tau.

FIGS. 19A-19B: Comparison of rhTau induced neurotoxicity towardnondifferentiated SH-SY5Y cells and retinoic-acid- (RA-) differentiatedSHSY5Y cells. The data combine toxicity results of 15.5nM monomeric,dimeric, and trimeric forms of both 1N4R and 2N4R tau variants. (a)After 3 hours-incubation, RA-differentiated SH-SY5Y cells are morevulnerable to extracellular trimeric rhTau toxicity thannondifferentiated SHSY-5Y cells are (P<0.05). (b) After 48-hoursincubation, nondifferentiated SH-SY5Y cells are more vulnerable toextracellular trimeric rhTau toxicity than RA-differentiated SH-SY5Ycells (P<0.05).

DETAILED DESCRIPTION OF THE INVENTION

Tau is a protein involved in microtubule function in the brain.Aggregation of tau can lead to neuronal damage and dementia andtraumatic brain injury. Increasing evidence suggests that small solubleoligomeric aggregate forms of tau may be the toxic species rather thanthe large fibrillar aggregates found during autopsies. Developingreagents against these species represents a potential therapeuticoption. In the present invention, using a bio-panning protocol toidentify single chain antibody fragments (scFv, also called nanobodies)against low (pico-molar) quantities of tau oligomers, the inventorsidentified binding reagents with therapeutic and diagnostic properties.Specifically, the inventors have generated single chain antibodyfragments (scFvs or nanobodies) that selectively recognize oligomericforms of the protein tau. These isolated scFvs that have potential valueas diagnostics, therapeutics and imaging agents for neurodegeneration.As diagnostics, these antibody fragments can be used to detect thepresence of oligomeric tau in serum, CSF or other fluid samples as apresymptomatic indication of neurodegeneration. Oligomeric tau may be anearly indicator of Alzheimer's disease, frontotemporal dementia, othertauopathies and of neurodegeneration following traumatic brain injury.The antibody fragments can also be used as therapeutics to selectivelytarget the toxic oligomeric tau aggregates protecting neurons fromdamage. Finally, the reagents can also be used as imaging agents todetect the presence of tau aggregates and neurodegeneration in vivo. Theantibody fragments can be readily labeled for PET scans or other imagingtechniques.

The biopanning studies were performed to isolate single chain variablefragments (nanobodies) against the different tau species. The biopanningprotocol that was used combines the imaging capabilities of AFM with thebinding diversity of phage-displayed antibody technology. To isolatenanobodies against specific oligomeric morphologies of a target protein,the protocol was modified to include negative panning steps to removeclones that bind to non-desired protein forms. To isolate nanobodiesagainst oligomeric tau two negative panning steps were incorporated. Inthe first negative panning step, all non-specific “sticky” clones wereremoved by panning against a generic protein, bovine serum albumin(BSA). In the second negative panning step, all clones that bind to thenon-desired monomeric form of tau were removed. A sample of puremonomeric tau was obtained for the negative panning to remove phageclones binding monomeric tau, and then aliquots of the remaining phagewere used to screen for dimeric and trimeric specific clonesrespectively. Since it was found that the trimeric tau species was muchmore toxic to human neuronal cell lines than monomeric or dimeric, theinventors focused efforts on isolating phage clones that were selectivefor trimeric tau 4N1R. After negative panning against BSA and monomerictau, ˜100 clones were obtained from the positive selection againsttrimeric tau 4N1R. Each phage clone was screened by AFM for binding tothe different tau species. Each phage sample was coincubated withmonomeric, dimeric and trimeric tau samples which had been previouslyfixed to a mica substrate. Unbound phage was removed by excess stringentrinsing and remaining bound phage were imaged by AFM. After screeningall 100 clones in this manner, clones that selectively bound eitherdimeric or trimeric tau, but not monomeric tau, were identified. Afterscreening all 100 phage clones, 6 clones were selected for further studybased on highest specificity for trimeric tau.

The DNA sequence of each of the six clones was validated to ensure thata full length scFv was encoded. In each of the six cases a single basepair was missing at the beginning of the coding sequence. In order toproduce soluble scFv for further characterization, it was necessary tocorrect the frame shift to enable efficient expression of the scFv. DNAand amino acid sequences of the clones are shown in FIGS. 3-5.Specifically, the amino acid sequences of the 6 selected cloned scFvsare: F9T (SEQ ID NO:1), F9T (SEQ ID NO:9), D11C (SEQ ID NO:11), D4G (SEQID NO:13), G12C (SEQ ID NO:15), H2A (SEQ ID NO:17), or H7T (SEQ IDNO:19). DNA sequences are also included in FIGS. 3-5.

The corrected F9 clone, F9T, expressed at very high levels, purifiedreadily and maintained high specificity for oligomer tau over monomerictau and fibril tau in the phage form viewed by AFM, so this clone wasselected for further study. The D11 clone was also identified asselectively binding to trimeric but not monomeric tau. Both clones alsoselectively recognize tau aggregates in post-mortem human brain tissuecontaining tau tangles but not in age matched normal tissue, althoughwith slightly different reactivity profiles. Therefore both F9T and D11Cnanobodies have promise as therapeutics to block neuronal toxicityinduced by naturally occurring aggregates of tau following TBI.

In a broad sense the scFv compositions of the present invention (e.g.,the F9T and D11C) may be described as compounds that are tau bindingcompounds. These compounds may therefore be used in diagnostic as welltherapeutic applications and may be either administered to patients orused on patient tissue samples. In some embodiments, the compositions ofthe present invention may be used for in vivo imaging of tau, anddistinguish between neurological tissue with toxic tau forms and normalneurological tissue. As such the nanobody compositions of the inventionmay be used to detect and quantitate tau oligomers in diseasesincluding, for example, Alzheimer's Disease, frontotemporal dementia,other tauopathies and of neurodegeneration following traumatic braininjury. In another embodiment, the compounds may be used in thetreatment or prophylaxis of neurodegenerative disorders. Also providedherein are methods of allowing the compound to distribute into the braintissue, and imaging the brain tissue, wherein an increase in binding ofthe compound to the brain tissue compared to a normal control level ofbinding indicates that the mammal is suffering from or is at risk ofdeveloping a neurodegenerative disease, such as Alzheimer's Disease,frontotemporal dementia, other tauopathies or neurodegenerationfollowing traumatic brain injury.

The methods of the present invention are conducted to provide earlystage diagnosis of Alzheimer's Disease, frontotemporal dementia, othertauopathies or neurodegeneration following traumatic brain injury. Asexplained herein the nanobodies of the invention (e.g., F9T or D11C) areones that specifically recognize tau oligomers (e.g., trimeric tau).Thus, compositions comprising these antibodies and antibody fragmentsmay be used to identify the presence of tau oligomers in a biologicalsample from a patient to be tested for a tauopathy, such as Alzheimer'sdisease, wherein the presence of tau oligomers in the sample isindicative that the patient has or is likely to develop the tauopathy(e.g., Alzheimer's disease). In certain embodiments, the assay formatthat is used may be any assay format that typically employs antibodycompositions. Thus, for example, the biological sample may be examinedusing immunohistology techniques, ELISA, Western Blotting, and the like.

For purposes of the diagnostic methods of the invention, thecompositions of the invention (e.g., F9T or D11C) may be conjugated to adetecting reagent that facilitates detection of the scFv. For example,example, the detecting reagent may be a direct label or an indirectlabel. The labels can be directly attached to or incorporated into thedetection reagent by chemical or recombinant methods.

In one embodiment, a label is coupled to the scFv through a chemicallinker. Linker domains are typically polypeptide sequences, such as polygly sequences of between about 5 and 200 amino acids. In someembodiments, proline residues are incorporated into the linker toprevent the formation of significant secondary structural elements bythe linker. In certain embodiments, linkers are flexible amino acidsubsequences that are synthesized as part of a recombinant fusionprotein comprising the RNA recognition domain. In one embodiment, theflexible linker is an amino acid subsequence that includes a proline,such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about100. In other embodiments, a chemical linker is used to connectsynthetically or recombinantly produced recognition and labeling domainsubsequences. Such flexible linkers are known to persons of skill in theart. For example, poly(ethylene glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulfhydryl linkages, or heterofunctional linkages.

The detectable labels can be used in the assays of the present inventionto diagnose a neurodegenerative disease, such as Alzheimer's Disease,these labels are attached to the scFvs of the invention, can be primarylabels (where the label comprises an element that is detected directlyor that produces a directly detectable element) or secondary labels(where the detected label binds to a primary label, e.g., as is commonin immunological labeling). An introduction to labels, labelingprocedures and detection of labels is found in Polak and Van Noorden(1997) Introduction to Immunocytochemistry, 2nd ed., Springer Verlag,N.Y. and in Haugland (1996) Handbook of Fluorescent Probes and ResearchChemicals, a combined handbook and catalogue Published by MolecularProbes, Inc., Eugene, Oreg. Patents that described the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Exemplary labels that can be used include those that use: 1)chemiluminescence (using horseradish peroxidase and/or alkalinephosphatase with substrates that produce photons as breakdown productsas described above) with kits being available, e.g., from MolecularProbes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL;2) color production (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

Where the scFv-based compositions of the invention (e.g., F9T and D11C)are contemplated to be used in a clinical setting, the labels arepreferably non-radioactive and readily detected without the necessity ofsophisticated instrumentation. In certain embodiments, detection of thelabels will yield a visible signal that is immediately discernable uponvisual inspection. One example of detectable secondary labelingstrategies uses an antibody that recognizes tau oligomers in which theantibody is linked to an enzyme (typically by recombinant or covalentchemical bonding). The antibody is detected when the enzyme reacts withits substrate, producing a detectable product. In certain embodiments,enzymes that can be conjugated to detection reagents of the inventioninclude, e.g., β-galactosidase, luciferase, horse radish peroxidase, andalkaline phosphatase. The chemiluminescent substrate for luciferase isluciferin. One embodiment of a fluorescent substrate for β-galactosidaseis 4-methylumbelliferyl-β-D-galactoside. Embodiments of alkalinephosphatase substrates include p-nitrophenyl phosphate (pNPP), which isdetected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer,and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′ diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination is used to detectthe detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

As noted herein throughout the scFvs of the invention (e.g., F9T andD11C) are targeted specifically to tau oligomers that are characteristicof Alzheimer's Disease, frontotemporal dementia, other tauopathies orneurodegeneration following traumatic brain injury. As such, the scFvsof the invention also may be used to specifically target therapeuticcompositions to the sites of tau aggregation. In this embodiment, anytherapeutic agent typically used for the treatment of these tauopathies,such as Alzheimer's disease, may be conjugated to scFvs in order toachieve a targeted delivery of that therapeutic agent. Various drugs forthe treatment of AD are currently available as well as under study andregulatory consideration. The drugs generally fit into the broadcategories of cholinesterase inhibitors, muscarinic agonists,anti-oxidants or anti-inflammatories. Galantamine (Reminyl), tacrine(Cognex), selegiline, physostigmine, revistigmin, donepezil, (Aricept),rivastigminc (Exelon), metrifonate, milameline, xanomeline, saeluzole,acetyl-L-carnitine, idebenone, ENA-713, memric, quetiapine, neurestroland neuromidal are just some of the drugs proposed as therapeutic agentsfor AD that can be conjugated to the scFv compositions of the inventionand targeted for therapeutic intervention of AD.

The scFv compositions of the invention can be used in any diagnosticassay format to determine the presence of tau oligomers. A variety ofimmunodetection methods are contemplated for this embodiment. Suchimmunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot, though several others are well known to those of ordinaryskill. The steps of various useful immunodetection methods have beendescribed in the scientific literature.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, polypeptide and/or peptide (in thiscase the tau oligomers), and contacting the sample with a firstantibody, monoclonal or polyclonal (in this case a scFv of theinvention, such as F9T or D11C), in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of immunocomplexes.

The immunobinding methods include methods for detecting and quantifyingthe amount of the tau oligomer component in a sample and the detectionand quantification of any immune complexes formed during the bindingprocess. Here, one would obtain a sample suspected of containing tauoligomers, and contact the sample with an antibody fragment of theinvention, such as F9T or D11C, and then detect and quantify the amountof immune complexes formed under the specific conditions.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those scFv molecules specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

As noted above, an scFv of the invention may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the scFV (e.g., F9T or D11C) is used to formsecondary immune complexes, as described above. After washing, thesecondary immune complexes are contacted with a third binding ligand orantibody that has binding affinity for the second antibody, again undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if this is desired.

One method of immuno detection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody (in the present example a scFv of the invention,such as F9T or D11C) is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed nanobody. In this method the sample to be tested is firstincubated in a solution containing the first step nanobody. If thetarget antigen is present, some of the nanobody binds to the antigen toform a biotinylated nanobody/antigen complex. The nanobody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to thenanobody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

The diagnostic assay format that may be used in the present inventioncould take any conventional format such as ELISA or other platforms suchas luminex or biosensors. The present invention shows the sequence ofthe F9T (SEQ ID NO:1), F9T (SEQ ID NO:9), D11C (SEQ ID NO:11), D4G (SEQID NO:13), G12C (SEQ ID NO:15), H2A (SEQ ID NO:17), or H7T (SEQ IDNO:19) scFvs. These sequences can readily be modified to facilitatediagnostic assays, for example a tag (such as GFP) can be added to thesescFvs to increase sensitivity. In one exemplary ELISA, antibodies (inthe present case the scFvs of the invention, such as F9T or D11C) areimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a test compositionsuspected of containing tau oligomers, such as a clinical sample (e.g.,a biological sample obtained from the subject), is added to the wells.After binding and/or washing to remove non-specifically bound immunecomplexes, the bound antigen may be detected. Detection is generallyachieved by the addition of another antibody that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA.”Detection may also be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withbinding agents (e.g., scFvs of the invention, such as F9T or D11C).After binding and/or washing to remove non-specifically bound immunecomplexes, the bound anti-binding agents are detected. Where the initialbinding agents are linked to a detectable label, the immune complexesmay be detected directly. Again, the immune complexes may be detectedusing a second antibody that has binding affinity for the first bindingagents, with the second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodies(or nanobodies) against an antigen are added to the wells, allowed tobind, and/or detected by means of their label. The amount of an antigenin an unknown sample is then determined by mixing the sample with thelabeled antibodies against the antigen during incubation with coatedwells. The presence of an antigen in the sample acts to reduce theamount of antibody against the antigen available for binding to the welland thus reduces the ultimate signal. This is also appropriate fordetecting antibodies against an antigen in an unknown sample, where theunlabeled antibodies bind to the antigen-coated wells and also reducesthe amount of antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.

In coating a plate with either tau oligomers or an scFv of the invention(e.g., F9T or D11C), one will generally incubate the wells of the platewith a solution of the antigen or scFvs, either overnight or for aspecified period of hours. The wells of the plate will then be washed toremove incompletely adsorbed material. Any remaining available surfacesof the wells are then “coated” with a nonspecific protein that isantigenically neutral with regard to the test antisera. These includebovine serum albumin (BSA), casein or solutions of milk powder. Thecoating allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting the tauoligomers and/or scFv composition with solutions such as BSA, bovinegamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. Theseadded agents also tend to assist in the reduction of nonspecificbackground.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

In various aspects of the invention, it will be desirable to furthersubject patients to more traditional diagnostic approaches fortauopathies, such as AD. Such general approaches for diagnosis are setout below.

The diagnosis of both early (mild) cognitive impairment and AD are basedprimarily on clinical judgment. However, a variety of neuropsychologicaltests aid the clinician in reaching a diagnosis. Early detection of onlymemory deficits may be helpful in suggesting early signs of AD, sinceother dementias may present with memory deficits and other signs.Cognitive performance tests that assess early global cognitivedysfunction are useful, as well as measures of working memory, episodicmemory, semantic memory, perceptual speed and visuospatial ability.These tests can be administered clinically, alone or in combination.Examples of cognitive tests according to cognitive domain are shown asexamples, and include “Digits Backward” and “Symbol Digit” (Attention),“Word List Recall” and “Word List Recognition” (Memory), “Boston Naming”and “Category Fluency” (Language), “MMSE 1-10” (Orientation), and “LineOrientation” (Visuospatial). Thus, neuropsychological tests andeducation-adjusted ratings are assessed in combination with data oneffort, education, occupation, and motor and sensory deficits. Sincethere are no consensus criteria to clinically diagnose mild cognitiveimpairment, various combinations of the above plus the clinicalexamination by an experienced neuropsychologist or neurologist are keyto proper diagnosis. As the disease becomes more manifest (i.e., becomesa dementia rather than mild cognitive impairment), the clinician may usethe criteria for dementia and AD set out by the joint working group ofthe National Institute of Neurologic and Communicative Disorders andStroke/AD and Related Disorders Association (NINCDS/ADRDA). On occasion,a clinician may request a head computed tomography (CT) or a headmagnetic resonance imaging (MRI) to assess degree of lobar atrophy,although this is not a requirement for the clinical diagnosis.

As noted above, there are various drugs that are presently in use orunder development for the treatment of Alzheimer's Disease,frontotemporal dementia, other tauopathies or neurodegenerationfollowing traumatic brain injury. The present invention contemplates theuse of scFvs of the invention, such as F9T or D11C, based “diagnostic”methods to further assess the efficacy of treatments. Given the role oftau in these diseases, the ability of a particular therapy to reduce theamount of oligomeric tau will be indicative of an effective treatment,as these forms have been shown to be toxic.

The present invention may involve the use of pharmaceutical compositionswhich comprise an agent conjugated to a scFv of the invention, such asF9T or D11C, for delivery into a subject having Alzheimer's disease,frontotemporal dementia, other tauopathies or neurodegenerationfollowing traumatic brain injury. Such an agent will ideally beformulated into a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, surfactants, antioxidants, preservatives(e.g., antibacterial agents, antifungal agents), isotonic agents,absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art. Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

A “variant” of an amino acid sequence of an antibody or antibodyfragment described herein or a nucleic acid sequence encoding such anamino acid sequence, is a sequence that is substantially similar to SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.Variant amino acid and nucleic acid sequences include syntheticallyderived amino acid and nucleic acid sequences, or recombinantly derivedamino acid or nucleic acid sequences. Generally, amino acid or nucleicacid sequence variants of the invention will have at least 40, 50, 60,to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generallyat least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

The present invention includes variants of the amino acid sequences ofthe antibodies and antibody fragments described herein, as well asvariants of the nucleic acid sequences encoding such amino acidsequences (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20 or SEQ ID NO:21). “Variants” are intended to include sequencesderived by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end, and/or addition ofone or more bases to the 5′ or 3′ end of the nucleic acid sequence;deletion or addition of one or more amino acids/nucleic acids at one ormore sites in the sequence; or substitution of one or more aminoacids/nucleic acids at one or more sites in the sequence. The antibodiesand antibody fragments described herein may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the enzyme can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. The substitution may bea conserved substitution. A “conserved substitution” is a substitutionof an amino acid with another amino acid having a similar side chain. Aconserved substitution would be a substitution with an amino acid thatmakes the smallest change possible in the charge of the amino acid orsize of the side chain of the amino acid (alternatively, in the size,charge or kind of chemical group within the side chain) such that theoverall enzyme retains its spatial conformation but has alteredbiological activity. For example, common conserved changes might be Aspto Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu andSer to Cys, Thr or Gly. Alanine is commonly used to substitute for otheramino acids. The 20 essential amino acids can be grouped as follows:alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophanand methionine having nonpolar side chains; glycine, serine, threonine,cystine, tyrosine, asparagine and glutamine having uncharged polar sidechains; aspartate and glutamate having acidic side chains; and lysine,arginine, and histidine having basic side chains.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

EXAMPLES Example 1

A vast number of studies have correlated protein aggregation withneurodegenerative diseases including AD, Parkinson's and Dementia withLewy Bodies. Numerous recent studies suggest that specific oligomericforms of these proteins are involved in neuronal toxicity and caninterfere with important functions including long term potentiation.Various soluble oligomeric species of Aβ and a-syn have been shown tooccur early during the course of AD and PD, and increasing evidenceimplicates oligomeric forms of tau in AD and other tauopathies.

Assays are being developed to study tau oligomer content in CSF, andinitial results suggest increased levels of tau oligomers in AD CSFcompared to non-AD specimens. We developed novel methods to purifyrecombinant human tau isoforms and to stabilize their oligomericstructures formed by disulfide linkages. Additionally, we purified taufrom human AD brain that retains its hyperphosphorylation. Thesepreparations have been used in mice to show that extracellular tauoligomers, but not monomer, inhibited long term potentiation ofhippocampal synapses and the formation of associative fear memory. Theoligomeric preparation of AD tau produced a similar effect indicatingthat hyperphosphorylation of tau did not affect inhibition of memory.Taken together tau oligomers were the forms of tau necessary to producethe disease-related effects and validate these structures as a targetfor drug discovery (Moe, J., et al. Validation of extracellular tauoligomer target for drug discovery in a novel animal model. in Societyfor Neuroscience. 2010. San Diego, Calif.).

We also developed a novel biopanning technology that combines theimaging capability of Atomic Force Microscopy (AFM) with the diversityof antibody libraries. This unique combination of antibody diversity andimaging capability has enabled us to isolate single chain antibodyvariable domain fragment (scFv or nanobody) reagents to an array ofmorphologies of key proteins involved in neurodegenerative diseasesincluding Aβ and alpha-synuclein (a-syn). We isolated nanobodies thatspecifically recognize monomeric (Emadi, S., et al., InhibitingAggregation of alpha-Synuclein with Human Single Chain AntibodyFragments. Biochemistry, 2004. 43: p. 2871-2878), fibrillar(Barkhordarian, H., et al., Isolating recombinant antibodies againstspecific protein morphologies using atomic force microscopy and phagedisplay technologies. Protein Eng Des Sel, 2006. 19: p. 497-502), andtwo different oligomeric a-syn morphologies (Emadi, S., et al.,Isolation of a human single chain antibody fragment against oligomericalpha-synuclein that inhibits aggregation and preventsalpha-synuclein-induced toxicity. J Mol Biol, 2007. 368: p. 1132-44;Emadi, S., et al., Detecting morphologically distinct oligomeric formsof alpha-synuclein. J Biol Chem, 2009. 284: p. 11048-58). Theanti-oligomeric a-syn nanobodies do not cross react with oligomeric Aβ,and specifically label PD brain tissue but not AD or healthy tissue(Emadi, S., et al., Detecting morphologically distinct oligomeric formsof alpha-synuclein. J Biol Chem, 2009. 284: p. 11048-58). In addition,we isolated nanobodies to different regions of full length Aβ (Liu, R.,et al., Single chain variable fragments against beta-amyloid (Abeta) caninhibit Abeta aggregation and prevent abeta-induced neurotoxicity.Biochemistry, 2004. 43: p. 6959-67) and to three distinct naturallyoccurring oligomeric Aβ morphologies (Zameer, A., et al.,Anti-oligomeric Abeta single-chain variable domain antibody blocksAbeta-induced toxicity against human neuroblastoma cells. J Mol Biol,2008. 384: p. 917-28). One, A4, specifically recognizes a largeroligomeric Ala species, inhibits aggregation and extracellular toxicityof Aβ, does not cross react with oligomeric a-syn, and specificallylabels Aβ aggregates in human AD brain samples, but not PD or healthybrain tissue (Zameer, A., et al., Anti-oligomeric Abeta single-chainvariable domain antibody blocks Abeta-induced toxicity against humanneuroblastoma cells. J Mol Biol, 2008. 384: p. 917-28). A secondnanobody, E1, recognizes a smaller trimeric or tetrameric Aβ species,and similar to A4 inhibits aggregation and extracellular toxicity of Aβ,does not cross react with oligomeric a-syn, and labels Aβ aggregates inhuman AD but not healthy brain tissue. Utilizing an AD brain derivedoligomeric Aβ preparation obtained from Dr. Selkoe (Walsh, D. M., etal., Naturally secreted oligomers of amyloid beta protein potentlyinhibit hippocampal long-term potentiation in vivo. Nature, 2002. 416:p. 535-9; Walsh, D. M. and D. J. Selkoe, Abeta Oligomers—a decade ofdiscovery. J Neurochem, 2007), we isolated a third nanobody, C6, thatspecifically recognizes oligomeric Aβ species derived from human ADbrain tissue, but does not recognize Aβ aggregates generated in vitro.The different specificities of each nanobody can be readily observedwhen each nanobody is expressed on the surface of a filamentousbacteriophage and antibody/antigen complexes are imaged by AFM(Kasturirangan, S., et al., Nanobody specific for oligomericbeta-amyloid stabilizes non-toxic form. Neurobiol Aging, 2010.).Therefore, the combination of antibody libraries and AFM imagingtechnologies enables us to isolate and carefully characterize reagentsthat recognize specific protein variants including four differentnaturally occurring aggregated forms of a-syn and four differentnaturally occurring aggregated forms of Aβ.

Another powerful advantage of our AFM panning protocol is that not onlycan we isolate and characterize reagents to specific proteinmorphologies, but we can do so using only picograms or less of material.In addition the sample does not need to be purified, and the proteindoes not need to be chemically modified in any way. We can actuallyisolate nanobodies against a single molecule of the target antigen(Shlyakhtenko, L. S., et al., Single-molecule selection and recovery ofstructure-specific antibodies using atomic force microscopy.Nanomedicine, 2007. 3: p. 192-7). This unique combination ofcapabilities to isolate different tau isoforms and to generate andcharacterize reagents that specifically recognize individual proteinvariants provides us with the means to generate reagents thatspecifically recognize an array of different tau variants present inhuman AD brain.

While several reagents already exist that can recognize monomeric andphosphorylated tau, these reagents cannot distinguish between differentaggregated states of tau. Reagents that can detect specific forms of taucan provide very powerful tools to facilitate diagnosis of AD and othertauopathies and to follow progression of these diseases or to evaluatetherapeutic strategies. While many neurodegenerative diseases haveoverlapping clinical symptoms and cellular and biochemical mechanismssuch as an increase in inflammatory markers, and aggregation of similarproteins, the reagents we propose to develop here will have well definedspecificities and selectivities for selected tau forms and shouldfacilitate specific diagnoses of AD and other tauopathies. Incombination with other protein and morphology specific reagents againstAβ and a-syn species, these reagents can be used to detect the presenceof biomarkers which can readily detect and distinguish many relatedneurodegenerative diseases including AD, PD, FTD and LBD.

Isolation of Different Size Oligomeric Tau Species from Human AD BrainTissue.

An array of different tau isoforms and aggregated assemblies has beenshown to be critically important in AD and other tauopathies. Reagentsthat can specifically label different tau species are needed to clarifythe roles of the different forms. Soluble tau oligomers are purifiedusing immunoaffinity and size fractionation to isolate oligomers withdifferent numbers of subunits and isoform composition and multiplepost-translational modifications associated with AD such ashyperphosphorylation, truncation, nitrosylation, ubiquitination andglycation. The oligomers in certain embodiments are heterogeneouscontaining known tau associating proteins such as beta amyloid, ApoE andalpha-synuclein. Aberrant binding of tau with other proteins associatedwith AD is also anticipated to facilitate generating disease-specificmorphologies for nanobody selection. Further, nanobodies are generatedagainst the different tau variants and the nanobodies are used toidentify which forms of tau best distinguish AD from healthy tissue inbrain and CSF samples.

Methods. Preparation of brain derived oligomeric tau. Tau oligomers arepurified from normal and AD brain specimens with tau pathology acquiredfrom the New York Brain Bank (The Taub Institute, Columbia University).Ten grams of tissue will be used for each preparation using the methoddeveloped by Ivanovova et al. (Ivanovova, N., et al., High-yieldpurification of fetal tau preserving its structure and phosphorylationpattern. J Immunol Methods, 2008. 339: p. 17-22) with modifications toisolate different size species of tau oligomers. The advantages of thismethod include the preservation of tau phosphorylation, simplicity, andhigh purity of product. We have already used a modified version of thismethod to isolate tau from an AD brain specimen and have shown thatphosphorylation state is preserved. Immunoblot analysis of recombinanttau441 and Tau purified from AD brain showed specific interaction of tauphospho-epitope-specific antibodies against tyrosine 231 and 217 with ADtau but no reactivity with recombinant tau, whereas phospho-independentmonoclonal antibody HT7 against total tau interacts with bothrecombinant and AD tau.

Brain tissue is homogenized in cold 1% perchloric acid, incubated on ice20 min., and centrifuged at 15,000×g for 20 min. The cleared supernatantis concentrated and buffer exchanged into buffer (20 mM Tris-HCl pH 7.4,150 mM NaCl, 0.1% Tween 20) using Amicon centrifugal devices(Millipore). Gel and Immunoblot analysis of cleared supernatantindicated the presence of multiple size aggregates of tau with multipletypes of modifications causing the appearance of a smear with somepredominant bands consistent in size with dimer and trimer. Thesespecies were purified by non-denaturing methods described below.Treatment of the preparation with reductant results in a lowered amountof higher molecular weight species indicating that at least some of thetau oligomers were stabilized by disulfide bonds. The incubation timecan be varied to increase or decrease the content of higher orderoligomeric tau.

Purification and characterization of oligomeric tau. Affinitychromatography to purify oligomeric tau from other protein species isperformed using the monoclonal antibody HT-7 (Thermo Scientific). Wehave observed that the tau epitope recognized by HT-7 is available forbinding in tau oligomers and because it binds tau independently ofphosphorylation status. The antibody (6 mg) will be coupled to CNBractivated Sepharose according manufacturer's protocol and packed into aPoly-Prep column C10/10 (GE Healthcare). The brain extract is filteredat a flow rate of 0.2 ml/min, and unbound protein are washed off withbuffer. Tau is eluted with 0.1 M glycine pH 2.6 in 0.5 ml fractionswhich is neutralized with 50 μl 1 M Tris-HCl pH 9 prior to analysis bynon-reducing SDS-PAGE.

Tau oligomers will be characterized and fractionated using high-pressureliquid chromatography (Beckman Coulter System Gold 32Karat HPLC). Ahigh-resolution gel filtration column (Biosep-SEC-3000, Phenomenex) isused to resolve tau oligomer species ranging in size from monomer (45.9Daltons) to dodecamer (552 KD). The Biosep-SEC-3000 has an exclusionrange of 5 to 700 kDa under native conditions. The column is run using1× PBS buffer using isocratic conditions at RT, and a run time of 40minutes. BSA (68 kDa) migrates with a retention time of 24.8 minutesunder these conditions. It is expected that a range of oligomers (dimer,trimer, tetramer . . . dodecamer) are resolved using the column althoughdepending on the species present some overlap is expected. Non-reducingSDS-PAGE and immunoblots are used to analyze the tau content in thefractions. AFM is used to determine the size distribution of the tauoligomers in the fractions as shown (see FIG. 1).

Generation and Characterization of Nanobodies Against Specific BrainDerived Oligomeric Tau Forms Isolated from AD Brain Tissue.

We have developed protocols that enable us to readily isolate individualclones from phage display libraries that recognize specific proteinmorphologies. We have continued to refine our panning protocols tofacilitate isolation of reagents against targets that are available inlimited amounts, that cannot be purified or that are unstable. Isolationof nanobodies against monomeric, fibrillar and two different oligomericforms of a-syn, and monomeric, fibrillar and two different oligomericβ-amyloid species have previously been performed. Also, isolation of ananobody against a third distinct oligomeric beta-amyloid morphology hasbeen performed. We have now included additional negative panning stepsto remove nonspecific and undesired binding activities, so virtually allclones isolated after only a single round of panning specificallyrecognize the target antigen. Using this technique, we can isolatevarious morphology specific ligands using only nanograms of target. Wehave also developed AFM based protocols to characterize ligand binding(Wang, M. S., et al., Characterizing Antibody Specificity to DifferentProtein Morphologies by AFM. Langmuir, 2008), so we can not only isolatemorphology specific ligands with only minimal material, but we cancharacterize binding specificity with limited material as well. Thisunique capability is ideally suited to isolating ligands againstspecific protein morphologies as we have demonstrated.

We have performed similar panning protocols to isolate nanobodiesagainst different morphologies of tau generated by syntheticallycross-linking tau monomers. In studies we have isolated nanobodies thatspecifically recognize synthetic dimeric, but not monomeric or synthetictrimeric tau.

Methods. AFM panning against tau aggregates. Nanobodies to the brainderived tau aggregate morphologies are isolated as described previously(Barkhordarian, H., et al., Isolating recombinant antibodies againstspecific protein morphologies using atomic force microscopy and phagedisplay technologies. Protein Eng Des Sel, 2006. 19: p. 497-502; Emadi,S., et al., Isolation of a human single chain antibody fragment againstoligomeric alpha-synuclein that inhibits aggregation and preventsalpha-synuclein-induced toxicity. J Mol Biol, 2007. 368: p. 1132-44;Emadi, S., et al., Detecting morphologically distinct oligomeric formsof alpha-synuclein. J Biol Chem, 2009. 284: p. 11048-58; Zameer, A., etal., Anti-oligomeric Abeta single-chain variable domain antibody blocksAbeta-induced toxicity against human neuroblastoma cells. J Mol Biol,2008. 384: p. 917-28). Only nanograms of material are required for thepresent panning protocols. To ensure that the nanobodies isolated fromthe panning protocol recognize oligomeric tau, a series of negativeselections is performed prior to the positive selection on the brainderived oligomeric tau samples. First a negative panning step isperformed on the control protein BSA to remove all non-specific stickynanobodies. Next a negative panning step is performed using brainderived monomeric tau to remove all nanobodies binding monomeric tau.Then a negative panning step is performed using a control non-diseasedbrain sample that was prepared similarly to the AD brain sample toremove nanobodies binding to non-disease associated forms of tau(primarily different monomeric forms) and any brain proteins that maypurify with tau. Verification is performed after each negative panningstep that all phage binding to the non-target samples have been removedby AFM. An aliquot of the remaining phage is added to mica containing afresh aliquot of the non-target sample and unbound phage are removed. Ifany phage are observed still binding to the off target samples, a secondround of negative panning is performed. The process is repeated until noremaining phage bind the off target sample. After the negative panningsteps, the remaining phage are added to the positive brain derived tauoligomer sample and positive clones recovered as described(Barkhordarian, H., et al., Isolating recombinant antibodies againstspecific protein morphologies using atomic force microscopy and phagedisplay technologies. Protein Eng Des Sel, 2006. 19: p. 497-502; Emadi,S., et al., Isolation of a human single chain antibody fragment againstoligomeric alpha-synuclein that inhibits aggregation and preventsalpha-synuclein-induced toxicity. J Mol Biol, 2007. 368: p. 1132-44;Emadi, S., et al., Detecting morphologically distinct oligomeric formsof alpha-synuclein. J Biol Chem, 2009. 284: p. 11048-58; Zameer, A., etal., Anti-oligomeric Abeta single-chain variable domain antibody blocksAbeta-induced toxicity against human neuroblastoma cells. J Mol Biol,2008. 384: p. 917-28).

Nanobody Characterization. There are numerous techniques that can beused to determine binding specificity of each of the nanobodies isolatedagainst the different target tau morphologies depending on theavailability and stability of the target antigen.

Specificity using Biacore, ELISA, Western Blot or Dot Blot. For thosetau morphologies that can be obtained in reasonable quantity, accuratebinding kinetics can be determined by surface plasmon resonance using aBIAcore X biosensor. Since chemical immobilization may affect variousaggregated protein morphologies, binding specificity can be determinedby ELISA, western or dot blot, depending on how easy it is to purify thetarget aggregate morphology. The protocols for each of these assays havebeen published (Emadi, S., et al., Isolation of a human single chainantibody fragment against oligomeric alpha-synuclein that inhibitsaggregation and prevents alpha-synuclein-induced toxicity. J Mol Biol,2007. 368: p. 1132-44; Emadi, S., et al., Inhibiting Aggregation ofalpha-Synuclein with Human Single Chain Antibody Fragments.Biochemistry, 2004. 43: p. 2871-2878; Zameer, A., et al., Single ChainFv Antibodies against the 25-35 Abeta Fragment Inhibit Aggregation andToxicity of Abeta42. Biochemistry, 2006. 45: p. 11532-9; Liu, R., etal., Single chain variable fragments against beta-amyloid (Abeta) caninhibit Abeta aggregation and prevent abeta-induced neurotoxicity.Biochemistry, 2004. 43: p. 6959-67; Zhou, C., et al., A humansingle-chain Fv intrabody blocks aberrant cellular effects ofoverexpressed alpha-synuclein. Mol Ther, 2004. 10: p. 1023-31; Liu, R.,et al., Residues 17-20 and 30-35 of beta-amyloid play critical roles inaggregation. J Neurosci Res, 2004. 75: p. 162-71; Liu, R., et al.,Proteolytic antibody light chains alter beta-amyloid aggregation andprevent cytotoxicity. Biochemistry, 2004. 43: p. 9999-10007).

Specificity using AFM If a oligomeric tau sample is not be able todetermined for nanobody specificity by conventional means such aswestern blot as described above, different AFM based methods can be usedto determine antibody specificity for antigen targets that are notsuitable for analysis as described above, or that are available in onlylimited amounts. Nanobody specificity can be determined by heightdistribution analysis as described (Wang, M. S., et al., CharacterizingAntibody Specificity to Different Protein Morphologies by AFM. Langmuir,2008), by recognition imaging (Marcus, W. D., et al., Isolation of anscFv targeting BRG1 using phage display with characterization by AFM.Biochem Biophys Res Commun, 2006. 342: p. 1123-9), or by using phagedisplayed nanobodies (see FIG. 1).

Identification of oligomeric tau specific nanobodies that distinguishbetween CSF and brain tissue from healthy and AD patients.

Individual nanobodies are screened against normal and AD brain tissuespecimens using ELISA, dot blot and immunohistochemistry to identifythose nanobody reagents that have the most potential to distinguishbetween healthy and AD cases.

Methods: Western- and Dot-blot assays: All assays are performedessentially as described (Emadi, S., et al., Isolation of a human singlechain antibody fragment against oligomeric alpha-synuclein that inhibitsaggregation and prevents alpha-synuclein-induced toxicity. J Mol Biol,2007. 368: p. 1132-44; Emadi, S., et al., Detecting morphologicallydistinct oligomeric forms of alpha-synuclein. J Biol Chem, 2009. 284: p.11048-58).

Immunohistochemistry of brain tissue. Brain tissue is pre-treated with0.1% triton X-100 for 30 minutes. Nanobody is then added (0.2 mg/ml) tothe brain sections and incubated for 1 hour at room temperature. Primaryantibodies (mouse anti-c-Myc and anti-Synaptophysin (Santa Cruz) 1/500dilution in BSA 3%) are then applied and incubated for 1 hour at roomtemperature. The brain sections are washed 3 times with PBS andincubated with 1/1000 dilution of secondary antibody in BSA 3% (goatanti-mouse IgG Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594,invitrogen) for 45 min at room temperature. Images are taken with aconfocal microscope at 60× magnification.

Example 2

Alzheimer's disease (AD) is a prevalent neurodegenerative disease inwhich the progressive neuronal loss and cognitive dysfunction areobserved. About 5 million Americans are living with AD and this numberis believed to triple by 2050. Unfortunately, no cure has been found asof yet. The main pharmacological approaches are symptom treatment suchas acetylcholine inhibitor. AD has two neuropathological features:extracellular deposit of amyloid beta fibrillar or diffuse andintracellular neurofibrillary tangle (NFT) of tau. Increasing evidencesuggests that protein misfolding, aggregation and fibril formation bothfeatures are closely associated with the pathogenesis of AD. Normal tauplays important role in assembling neuron microtubule and stabilizingits structure and physiological function while abnormallyhyperphosphorylated tau and its oligomeric and aggregated forms areconsidered correlated with synapse loss. Reagents targeting oligomerictau over monomeric or aggregated tau or any nonspecific protein thatpotentially interfere with the aggregation process without disturbingthe normal tau function are needed. Besides, detecting tau oligomer notaggregated tau with such reagents is a promising diagnostic approach inearly stage of AD cases.

As described herein, single chain variable fragment (scFv) against tauoligomers is such a reagent. scFv is a fusion of one pair of heavy chainand light chain variable domains of immunoglobulin G (IgG) to make oneantigen-binding site which is specific to only one epitope on theantigen. scFv specificity can be increased by affinity mature. scFv hassmaller molecular weight (291d)) potentially penetratingblood-brain-barrier before it's compromised in the later stage of AD.Due to lack of constant domains, scFv is unlikely to induce inflammationin clinical test. To fulfill scFv screening, recovering and reproducing,we used Sheets phagemid library which is a human phage-displayed scFvlibrary of up to 6.7×10⁹ variety. An individual phage-display scFv cloneis a filamentous bacteriophage with a molecule of scFv expressed on itssurface and linked with a g3p. It is easy to be identified with atomicforce microscopy (AFM) and infectious to common E. coli strains tofacilitate genetic modification.

We have successfully performed a novel biopanning combiningphage-displayed library and AFM to obtain scFv clones specific totrimeric tau which shows in Lactate Dehydrogenase (LDH) test as the mosttoxic of all available tau species in our lab. After DNA sequencemodification, F9T keeps specificity to oligomeric tau and displaysefficient soluble scFv expression and purification. F9T alsodemonstrated potentials of discriminate AD from ND on human middletemporal gyrus (MTG) tissue and human cerebrospinal fluid, both of whichare enriched with abnormal tau in AD.

Results.

I. Select scFv Against Different Tau Oligomeric Forms Using Purified TauDimer, Trimer, Mixed Oligomer and Monomer Samples Using AFM BiopanningProtocols

A. Select the Most Toxic Morphology of Tau Through LDH Test on SH-SYSYNeuroblastoma Cell Line and its Cholinergic Differentiated Form.

In order to identify the most promising oligomeric tau species to targetwith our antibody fragment library, we first tested which tau specieswere toxic toward a neuronal cell line. We performed cell viabilityassays using undifferentiated and cholinergic like SH-SY5Y humanneuroblastoma cells treated with monomeric, dimeric or trimeric tau. Wetested two different isoforms of tau, 4N1R and 4N2R and measuredtoxicity using an LDH assay as described. As shown in FIG. 2(A) and (B),trimeric tau 4N1R and 4N2R were toxic to undifferentiated SHSY-5Y cellswhile monomeric and dimeric tau were not. Essentially identical resultswere obtained when the SHSY-5Y cells were first differentiated to acholinergic-like phenotype (data not shown) before treatment withmonomeric, dimeric or trimeric tau. Toxicity induced by trimeric taushowed concentration dependence. These results suggest that trimeric taumay be a critically important species in the progression of AD.

B. Biopanning Against Purified Synthetic Trimeric Tau 4N1R Using theSheets Phagemid Library.

We performed biopanning studies to isolate single chain variablefragments (nanobodies) against the different tau species. We utilize anovel biopanning protocol that combines the imaging capabilities of AFMwith the binding diversity of phage-displayed antibody technology. Toisolate nanobodies against specific oligomeric morphologies of a targetprotein, we have modified the protocol to include negative panning stepsto remove clones that bind to non-desired protein forms. To isolatenanobodies against oligomeric tau we incorporated two negative panningsteps. In the first negative panning step, we remove all non-specific“sticky” clones by panning against a generic protein, bovine serumalbumin (BSA). In the second negative panning step, we remove all clonesthat bind to the non-desired monomeric form of tau. During our negativepanning, we removed as many non-desired clones as possible. The purityof monomeric tau was critical since we did not want to lose oligomerictau clones during this step. We obtained a sample of pure monomeric taufor the negative panning to remove phage clones binding monomeric tau,and then used aliquots of the remaining phage to screen for dimeric andtrimeric specific clones respectively. Since we found that the trimerictau species was much more toxic to human neuronal cell lines thanmonomeric or dimeric, we focused our efforts on isolating phage clonesthat were selective for trimeric tau 4N1R.

C. Screen Monoclonal Phage Specific to Trimeric Tau 4N1R by AFM

After negative panning against BSA and monomeric tau, we recoveredaround 100 clones from the positive selection against trimeric tau 4N1R.Since the amounts of tau samples provided to us by Oligomerix waslimiting, we were not able to screen each of the phage by conventionalELISA to determine which of the 100 clones had high specificities fortrimeric over monomeric tau. Instead we screened each individual phageclone by AFM for binding to the different tau species. We coincubatedeach phage sample with monomeric, dimeric and trimeric tau samples whichhad been previously fixed to a mica substrate. Unbound phage was removedby excess stringent rinsing and remaining bound phage were imaged byAFM. After screening all 100 clones in this manner, we identified clonesthat selectively bound either dimeric or trimeric tau, but not monomerictau. The AFM panning protocol allows us to screen all 100 clones forbinding specificity using only nanogram amounts of antigen, although theassay is quite time-consuming. After screening all 100 phage clones, weselected 6 clones for further study based on highest specificity fortrimeric tau.

D. DNA Sequencing and Frame-Shift Correction of Isolated Clones.

We validated the DNA sequence of each of the six clones to ensure that afull length scFv was encoded (FIG. 4). In each of the six cases a singlebase pair was missing at the beginning of the coding sequence. In orderto produce soluble scFv for further characterization, we needed tocorrect the frame shift to enable efficient expression of the scFv. Wedesigned forward and reverse primers which enabled us to modify eachscFv sequence by polymerase chain reaction (PCR) and correct the frameshift (see, e.g., FIG. 3). We then cloned the corrected scFv sequenceinto an expression plasmid that also contained a c-myc tag foridentification and a poly-histidine tag for purification. The correctedF9 clone, F9T, expressed at very high levels, purified readily andmaintained high specificity for oligomer tau over monomeric tau andfibril tau in the phage form viewed by AFM, so this clone was selectedfor further study.

II. Select Fragments that Show Disease Specificity in AD Brain Sections

A. Preliminary Specificity and Affinity Test of Selected Nanobodies onAge Matched Alzheimer's and Non-Demented Brain Sections

While monomeric tau plays a crucial role in microtubule assembly andstability, oligomeric tau is toxic to cells. Oligomeric tau may be theresult of tau hyperphosphorylation and other posttraslationalmodifications. Oligomeric tau dettaches from microtubules and may thenaggregate further to form the neurofibillary tangles which are ahallmark feature of Alzheimer's. It is likely that misfolding andaggregation of tau is intimately linked with misfolding and aggregationof amyloid-beta (Aβ), so detection of the different oligomeric forms ofthese proteins has promise in diagnosis and treatment of Alzheimer's. Weanalyzed the reactivity of the F9T nanobody against trimeric tau withhomogenized post-mortem brain tissue, which was obtained from the middletemporal gyrus of different Braak stage Alzheimer's brain defined by theextraneuronal plaque frequency. All brain samples are of age matched andwere generously provided by Thomas G. Beach from Banner Sun HealthResearch Institute. We analyzed six samples obtained from non-dementedsources (ND1 to ND6). The ND samples, patients who demonstrated noobvious symptons of dementia, were broken into two categories based onpresence of Aβ plaques: ND1, ND2 and ND3 had no Aβ plaque (Braak stage Ito II) while ND4, ND5 and ND6 all had slight plaque frequency (Braakstage Ito II). The six Alzheimer's brain tissue samples (AD1 to AD6)were from patients diagnosed with Alzheimer's disease. The samples weredivided by plaque frequency where AD1, AD2 and AD3 brain samples showmoderate frequent plaque (Braak stage III to IV) while AD4, AD5 and AD6brain samples show the most severe plaque frequency (Braak stage III toIV). Both F9T preparations show similar reactivity where the strongestsignals are obtained from AD2 and AD3, although strong signals areobtained with 5 of 6 AD samples. Interestingly, there is almost noreactivity with any of the cognitively normal tissue samples that didnot contain any Aβ plaques. The three cognitively normal samples thatdid contain plaques did show reactivity suggesting an interactionbetween Aβ aggregation and tau aggregation as noted above. Anotherinteresting trend is that the AD1-3 samples have higher reactivity onaverage than the AD4-6 samples. The high plaque frequency of th AD4-6samples may indicate the presence of more neurofibrillar tau and lessoligomeric tau.

B. Preliminary Specificity Test of Selected Nanobody Displayed Phage F9Ton Human Cerebrospinal Fluid (CSF) as a Potential Diagnostic Technique

Total tau (T-tau) and phosphorylated tau (P-tau) levels in CSF areimportant biomarkers for Alzheimer's for several reasons. That T-taulevel increase in AD CSF can discriminate sporadic AD from non-dementedage-matched controls with high sensitivity. T-tau level also reflectsneuronal and axonal degeneration which enables broader use to otherdementias than AD such as Creutzfeldt-Jakob disease (CJD) and Lewy bodydisease (LBD). Compared with normal level of P-tau in other commondementias and normal aging, P-tau level increase markedly in AD.Combining this feature with decreased Aβ42 in CSF, a promisingdiagnostic approach can be obtained. F9T's ability of recognizing tauover other forms of amyloid can be a crucial part of such an approach.F9T nanobody displayed phage's interaction with human CSF proteins wasimaged by atomic force microscopy. The preliminary result demonstratesthe presence of phage binding in AD versus the absence of phage in ND,which is in accordance with the fact that AD CSF contains an increasedlevel of total tau. While parameters may be adjusted, this test needs ispotentially a more sensitive way of detecting CSF tau level and apromising diagnostic technique for AD and other tauopathies.

Example 3

Traumatic Brain Injury (TBI) affects over 1.7 million people each year,and over 230,000 soldiers have suffered TBI on the battlefield(http://www.dvbic.org/traumatic-brain-injury-tbi-awareness-and-prevention).Around 10-20% of soldiers serving in Iraq and Afghanistan have sufferedTBI from different sources. It is well established that traumatic braininjury (TBI) can disrupt cognitive functioning. The brain is verysensitive to stress and injury and responds by expressing a variety ofneuromorphological and neurochemical changes. TBI induces axonal injuryand damage to protein transport mechanisms, so neurofilament proteins,such as tau, which accumulate in axons following TBI, play a criticalrole in TBI. Following TBI, increased levels of tau in brain fluid, CSFand serum samples are all predictive of adverse long-term clinicaloutcomes. Neurofibrillary tau aggregates have been identified insoldiers suffering from TBI as well as in many athletes such as footballplayers that suffer repeated head trauma, suggesting a similar mechanismbehind these injuries. Aggregates of tau are also the major component ofthe neurofibrillary tangles that are a hallmark feature in the brains ofAlzheimer's disease (AD) patients, and TBI is a risk factor for AD.Therefore tau clearly plays a critical role in brain function,particularly cognitive functions, and the ability of tau to supportcognitive function is impaired following TBI.

Mild traumatic brain injury (mTBI) frequently leads to chronic traumaticencephalopathy (CTE) and other neurodegenerative disorders including AD,Parkinson's disease (PD), and amyotrophic lateral sclerosis. While themechanism of progression and risk factors for mTBI to convert to CTE andother neurodegenerative diseases are not well known, accumulation of tauaggregates in neurofibrillary tangles (NFTs) and glial tangles invarious regions of the brain are a common feature indicating that tau isa viable therapeutic target to prevent neurodegeneration following TBI.Tau is a natively unfolded protein that can aberrantly fold into variousaggregate morphologies including β-sheet containing fibrillar formsfound in the NFTs and in different oligomeric species (Garcia-Sierra,F., et al., Conformational changes and truncation of tau protein duringtangle evolution in Alzheimer's disease. J Alzheimers Dis, 2003. 5: p.65-77; Ghoshal, N., et al., Tau conformational changes correspond toimpairments of episodic memory in mild cognitive impairment andAlzheimer's disease. Exp Neurol, 2002. 177: p. 475-93; Grundke-Iqbal,I., et al., Abnormal phosphorylation of the microtubule-associatedprotein tau (tau) in Alzheimer cytoskeletal pathology. Proc Natl AcadSci USA, 1986. 83: p. 4913-7; Schweers, O., et al., Structural studiesof tau protein and Alzheimer paired helical filaments show no evidencefor beta-structure. J Biol Chem, 1994. 269: p. 24290-7). While NFTs havebeen implicated in mediating neurodegeneration in AD and tauopathies,animal models of tauopathy have shown that memory impairment and neuronloss do not associate well with accumulation of NFT, but do associatewell with oligomeric forms of tau (Santacruz, K., et al., Tausuppression in a neurodegenerative mouse model improves memory function.Science, 2005. 309: p. 476-81) and a regional dissociation of neuronloss and NFT pathology. The pathological structures of tau most closelyassociated with AD progression were shown to be tau oligomers (Berger,Z., et al., Accumulation of pathological tau species and memory loss ina conditional model of tauopathy. J Neurosci, 2007. 27: p. 3650-62;Maeda, S., et al., Increased levels of granular tau oligomers: an earlysign of brain aging and Alzheimer's disease. Neurosci Res, 2006. 54: p.197-201; Sahara, N., S. Maeda, and A. Takashima, Tau oligomerization: arole for tau aggregation intermediates linked to neurodegeneration. CurrAlzheimer Res, 2008. 5: p. 591-8). Similar to the many studies thatimplicate oligomeric rather than fibrillar forms of Aβ in neuronaldysfunction, these studies all indicate that oligomeric tau aggregates,rather than tau tangles, are acutely neurotoxic and are responsible forthe neurodegenerative phenotype. Therefore toxic oligomeric tau speciesare likely play a critical role in neurodegeneration following TBI.Oligomeric tau species have been shown to contribute to neurotoxicitythrough an “infectious” model of disease progression. Extracellular tauaggregates can initiate tau misfolding intracellularly (Frost, B., R. L.Jacks, and M. I. Diamond, Propagation of tau misfolding from the outsideto the inside of a cell. J Biol Chem, 2009. 284: p. 12845-52), taupathology spreads contiguously throughout the brain from early to latestage disease (Schonheit, B., R. Zarski, and T. G. Ohm, Spatial andtemporal relationships between plaques and tangles inAlzheimer-pathology. Neurobiol Aging, 2004. 25: p. 697-711), and brainextract from a transgenic mouse with aggregated mutant human tautransmits tau pathology when introduced into the brains of miceexpressing normal human tau (Clavaguera, F., et al., Transmission andspreading of tauopathy in transgenic mouse brain. Nat Cell Biol, 2009.11: p. 909-13). A receptor-mediated mechanism for the spread of taupathology by extracellular tau has been described (Gomez-Ramos, A., etal., Characteristics and consequences of muscarinic receptor activationby tau protein. Eur Neuropsychopharmacol, 2009. 19: p. 708-17). Thesestudies further support oligomeric tau as a particularly promisingtherapeutic target for TBI-neurodegeneration.

The present inventors have developed unique technology that enables usto isolate reagents that bind specific morphologies of a target protein.The inventors have combined the imaging capabilities of atomic forcemicroscopy (AFM) with the binding diversity of phage display antibodytechnology to allow us to identify the presence of specific proteinmorphologies and then isolate reagents that bind a target morphology(Barkhordarian, H., et al., Isolating recombinant antibodies againstspecific protein morphologies using atomic force microscopy and phagedisplay technologies. Protein Eng Des Sel, 2006. 19: p. 497-502). Theinventors have developed a series of morphology specific single chainantibody fragments (nanobodies) that have great promise fordistinguishing between different neurodegenerative diseases and fortargeting specific toxic aggregate species and have recently isolatedseveral nanobodies that selectively bind toxic oligomeric tau species.The nanobodies distinguish between AD and healthy post-mortem humantissue and can detect oligomeric tau in post-mortem AD CSF samples. Herethe inventors utilize the oligomeric tau specific nanobodies astherapeutics to block toxicity of tau following TBI in mouse models. Thepool of morphology-specific nanobodies are also used to analyze braintissue of the mice for the presence of different aggregated proteinspecies.

Neurobehavioral, biochemical and neuropathological characterization ofmouse models of neurodegeneration (Abdullah, L., et al., Proteomic CNSprofile of delayed cognitive impairment in mice exposed to Gulf Waragents. Neuromolecular Med. 13: p. 275-88; Abdullah, L., et al.,Lipidomic Profiling of Phosphocholine Containing Brain Lipids in Micewith Sensorimotor Deficits and Anxiety-Like Features After Exposure toGulf War Agents. Neuromolecular Med; Todd Roach, J., et al., Behavioraleffects of CD40-CD40L pathway disruption in aged PSAPP mice. Brain Res,2004. 1015: p. 161-8), in particular different TBI models (Crawford, F.,et al., Identification of plasma biomarkers of TBI outcome usingproteomic approaches in an APOE mouse model. J Neurotrauma. 29: p.246-60; Crawford, F., et al., Apolipoprotein E-genotype dependenthippocampal and cortical responses to traumatic brain injury.Neuroscience, 2009. 159: p. 1349-62; Crawford, F. C., et al., Genomicanalysis of response to traumatic brain injury in a mouse model ofAlzheimer's disease (APPsw). Brain Res, 2007. 1185: p. 45-58; Ferguson,S., et al., Apolipoprotein E genotype and oxidative stress response totraumatic brain injury. Neuroscience. 168: p. 811-9) including arecently developed mild TBI (mTBI) model of single (s-mTBI) andrepetitive (r-mTBI, 5 injuries given at 48 h intervals) injury (Mouzon,B. C., et al., Repetitive mild traumatic brain injury in a mouse modelproduces learning and memory deficits accompanied by histologicalchanges. J Neurotrauma) have been previously published. In wild typeC57BL/6 mice this injury demonstrates acute motor and cognitive deficitsin both paradigms, but more significant deficits following r-mTBI, andneuropathological analyses show axonal injury and reactive gliosis, moreevident in the r-mTBI mice. Ongoing studies reveal progressiveneuropathological changes and persistence of neurobehavioral deficits inthe r-mTBI model at 6, 12 and 18 months, whereas s-mTBI recover thelevel of performance of anesthesia control groups. We have alsoadministered this mTBI paradigm to the hTau transgenic mouse whichexpresses all isoforms of non-mutant human tau on a null murine taubackground. In young hTau mice r-mTBI appears to precipitatehyperphosphorylation of Tau while in aged hTau mice (18 months) r-mTBIexacerbates the existing burden of Tau pathology and glial activation.This r-mTBI model in hTau mice is thus an ideal platform in which toevaluate potential TBI therapeutics targeting tau pathogenicity. Tauinduced toxicity and memory deficits following TBI can be safely reducedby selectively targeting toxic oligomeric tau species using recombinantantibody fragments that do not initiate an inflammatory response.

Example 4

Isolation and Characterization of Single Chain Variable FragmentsSelective for a Neurotoxic Oligomeric Tau Species

Alzheimer's disease (AD) is a devastating progressive neurodegenerativedisease that causes brain atrophy, memory deterioration and cognitveloss in affected individuals. It is the sixth leading cause of death inthe United States, currently affecting over 5.4 million Americans withannual costs of over $200 billion in medical care. Although AD was firstdiscovered over a hundred years ago, and substantial progress has beenmade in understanding the etiology of the disease, there are still noeffective therapeutic or definitive diagnostic approaches available. ADis characterized by the presence of two hallmark pathologies:extracellular neuritic plaques containing insoluble fibrillar aggregatesof amyloid-beta (Aβ) and intracellular neurofibrillary tangles (NFTs)containing fibrillar aggregates of tau. Although these insolubleaggregated species have long been considered as the primary toxicelements of AD, increasing evidence indicates that small solubleoligomeric forms of both Aβ and tau play more critical roles in theonset and progression of AD than the fibrillar aggregates. The role ofAβ aggregation in AD in particular has been extensively studied, howeverdespite very promising results in animal models, various therapeuticroutes of targeting Aβ aggregation have had only very limited success inclinical trials. In part due to the rather disappointing resultsobtained from therapeutic trials targeting Aβ, the role of tau in theprogression of AD is gaining more attention, including studies toelucidate the roles of different variants and aggregate forms of tau.

Tau is a microtubule-associated protein, generally located in the axonsof neurons, where it is involved in the assembly and stabilization ofmicrotubules from tubulin. Although human tau is encoded by a singlegene on chromosome 17q21, six major tau isoforms can be formed byalternative splicing of exons 2, 3 and 10. Tau can also bepost-translationally modified by phosphorylation, glycosylation,ubiquitinylation, or glycation among others resulting in a wide varietyof different tau species that can exist in vivo. Sincehyperphosphorylated tau species are predominantly found in the hallmarkNFTs, phosphorylation of tau has been extensively studied and inhibitingkinases involved in tau phosphorylation has been pursued as a potentialtherapeutic approach. Levels of hyperphosphorylated tau have also beenstudied as biomarkers for AD, and ratios of different tau isoformsparticularly phosphorylated variants correlate well with tauopathiesincluding FTD and AD. Hyperphosphorylation of the microtubule-bindingdomain (MBD) of tau results in a conformational change that promotesmisfolding and loss of physiological function. However, phosphorylationof tau may also be required for some cellular functions including adultneurogenesis, as new adult-born granule neurons contain a significantamount of a hyperphosphorylated three repeat tau variants. Therapeuticstrategies aimed at regulating kinase activity bear the risk ofinterrupting normal phosphorylation dependent functions of tau. Giventhe complexity of the many different potential isoforms of tau that canoccur in vivo and the uncertainty as to the physiological effects of tauhyperphosphorylation and aggregation, the roles of differenthyperphosphorylated and aggregated tau variants in AD remaincontroversial and the most promising diagnostic or therapeutic targetsare still not known.

Similar to the neurotoxic effects observed with soluble oligomericaggregates of Aβ, numerous studies indicate that soluble aggregates oftau play an important role in the pathology of AD. Both brain derivedand recombinant oligomeric tau aggregate species disrupt intracellularcalcium levels and are toxic to cultured human neuronal cells when addedextracellularly. In animal models expressing human tau,neurodegeneration-related phenotypes including behavioral impairments,neuronal loss, and synapse lesions correlate better with the presence ofsoluble tau oligomeric and prefilament species than with fibrillar NFTlevels. Neuronal loss also precedes NFTs formation suggestinginvolvement of other species such as oligomeric tau variants. Inpostmortem human brains, high oligomeric tau levels were detected in thefrontal lobe cortex at early stages of AD before the presence of NFTs.Oligomeric tau may also be responsible for transmission of pathologywith a prion-like mechanism as NFT tau pathology spreads from brainregions seeded with oligomeric tau into other regions resulting inaggregation of endogenous tau. We have previously shown usingnon-phosphorylated recombinant human tau (NPrhTau) that trimeric, butnot monomeric or dimeric aggregates are toxic to human neuronal cells.

Here we describe isolation of antibody based reagents that selectivelyrecognize the toxic NPrhTau trimeric species. We used a single chainvariable domain antibody fragment (scFv) library (Sheets, M. D., et al.,Efficient construction of a large nonimmune phage antibody library: theproduction of high-affinity human single-chain antibodies to proteinantigens. Proc Natl Acad Sci USA, 1998. 95(11): p. 6157-62) as source ofbinding diversity and an atomic force microscopy (AFM) based biopanningprotocol (Barkhordarian, H., et al., Isolating recombinant antibodiesagainst specific protein morphologies using atomic force microscopy andphage display technologies. Protein Eng Des Sel, 2006. 19(11): p.497-502; Emadi, S., et al., Isolation of a human single chain antibodyfragment against oligomeric alpha-synuclein that inhibits aggregationand prevents alpha-synuclein-induced toxicity. J Mol Biol, 2007. 368(4):p. 1132-44; Emadi, S., et al., Detecting morphologically distinctoligomeric forms of alpha-synuclein. J Biol Chem, 2009. 284(17): p.11048-58; Kasturirangan, S., et al., Nanobody specific for oligomericbeta-amyloid stabilizes nontoxic form. Neurobiol Aging, 2012. 33(7): p.1320-8; Kasturirangan, S., et al., Isolation and characterization ofantibody fragments selective for specific protein morphologies fromnanogram antigen samples. Biotechnol Prog, 2013. 29(2): p. 463-71;Zameer, A., et al., Single chain Fv antibodies against the 25-35 Abetafragment inhibit aggregation and toxicity of Abeta42. Biochemistry,2006. 45(38): p. 11532-9) as a selection tool to isolate scFvs thatselectively bound the trimeric tau species. We utilized severalsubtractive panning steps in the selection protocol to ensure theremoval of all scFvs cross-reactive with monomeric tau and otheroff-target proteins. We identified three different scFvs that boundtrimeric but not monomeric or fibrillar tau. The three different scFvsall bound distinct epitopes on the trimeric tau aggregate. The scFvsreacted with naturally occurring oligomeric tau in brain tissue from atransgenic AD mouse model that overexpresses both Aβ and tau and showedthat significant levels of oligomeric tau are present in brain tissuefrom this mouse model long before NFTs are detected. The scFvs alsoreacted with oligomeric tau naturally present in post-mortem human ADbrain tissue. Levels of oligomeric tau in the post-mortem human braintissue correlated with progression of AD as oligomeric tau levelsincrease with Braak stage.

Materials and Methods

scFv Phage Display Library—The Sheets phage display scFv library(Sheets, M. D., et al., Efficient construction of a large nonimmunephage antibody library: the production of high-affinity humansingle-chain antibodies to protein antigens. Proc Natl Acad Sci USA,1998. 95(11): p. 6157-62) with an estimated diversity of 6.7×10⁹ wasgenerously provided by Dr. Yu Zhou (Department of Anesthesia, Universityof San Francisco) and used for biopanning. Phage was produced andpurified as previously described (Marks, J. D., et al., By-passingimmunization. Human antibodies from V-gene libraries displayed on phage.J Mol Biol, 1991. 222(3): p. 581-97). A final titre of 10¹³-10¹⁴ pfu/mLwas used for biopanning.

Aggregated Tau species—Two isoforms (1N4R and 2N4R) ofnon-phosphorylated recombinant human tau (NPrhTau) species were used forthe panning protocols. Monomeric and oligomeric forms of tau weregenerated as described above. A fibrillar 2N4R aggregate stock wasprepared following a heparin fibrillation protocol by mixing rhTau 2N4Rmonomer (final molarity of 4 μM) and low molecular weight heparin (finalmolarity of 4 μM) in final 20 mM tris-HCl pH 7.4 and final 5 mMDL-Dithiothreitol (DTT) in deionized water (DI water). The mixture wasincubated at 37° C. for 2 weeks with occasional stirring.

Biopanning against NPrhTau 1N4R trimer—The biopanning process wasperformed essentially as previously described (Kasturirangan, S., etal., Isolation and characterization of antibody fragments selective forspecific protein morphologies from nanogram antigen samples. BiotechnolProg, 2013. 29(2): p. 463-71) with the following modifications. Thebiopanning process is divided into “subtractive panning” and “positivepanning” steps (FIG. 6). The subtractive panning steps are designed toremove all scFv-displayed phage from the library pool which bind tonon-desired antigens including a control protein bovine serum albumin(BSA) used to remove non-specific binding phage and monomeric tau usedto remove all phage binding non-aggregated linear epitopes of tau. Thepositive panning step then recovers any scFv-displayed phage from theremaining phage pool that selectively bind trimeric tau. Each step ismonitored by AFM to ensure that essentially all phage binding BSA andmonomeric tau are removed and phage binding to trimeric tau arerecovered.

Subtractive panning step—A set of high affinity immunotubes were coatedwith 2 mL/tube of 1 mg/mL BSA in carbonate/bicarbonate coating buffer pH9.6 and another set with 2 mL of 12 μg/mL tau 1N4R monomer in the samecoating buffer and incubated overnight at 4° C. After antigenimmobilization, immunotubes were washed extensively with phosphatebuffered saline (PBS) and sealed to keep moist. A total volume of 0.5 mLof the phage display library was added to the first tube coated withBSA. The tube was then sealed and incubated at room temperature for 30minutes with gentle agitation ensuring that the phage solution did notcontact uncoated regions in the immunotube. After incubation, the phagesolution was removed and additional unbound phage rinsed off with 100 μlPBS. The phage and rinse solutions were combined and added to a secondtube containing BSA and then to sequential tubes following the sameprocedure for each tube. The final recovered phage solution volume wasapproximately 1 mL. A 10 μL aliquot of phage solution recovered afterincubation with each tube was added to mica containing BSA and imaged byAFM to determine whether there were any phage remaining in the phagepool that could still bind BSA. If no bound phage were observed, thesubtractive panning step successfully removed essentially all phagebinding to the target antigen, in this case BSA. A second subtractivepanning round was performed using immunotubes coated with monomeric 1N4RrhTau to remove all phage binding monomeric tau. The process wasperformed and monitored as described above. After the second round ofsubtractive panning, the final remaining phage solution was stored in100 μL aliquots at −80° C.

Positive panning—A 10 μl aliquot of 60 μg/mL of positive target antigen,trimeric rhTau 1N4R, was deposited on a piece of freshly cleaved mica,incubated at room temperature for 10 minutes, and then extensivelywashed with DI water and dried. A 200 μl aliquot of the remaining phagepool obtained after subtractive panning was added to the mica, incubatedat room temperature for 10 minutes, and then washed with 2 mL 0.1%tween/DI water and at least 10 mL DI water to remove all unbound phage.The positive panning step was performed in duplicate for analysis by AFMto verify the presence of phage binding trimeric tau. Bound phage wereeluted with 1.4% triethylamine (TEA) and neutralized after 5 minuteswith an equal volume of 1M Tris-HCl pH 7.4 buffer. The eluted phagestock were recovered as described (Emadi, S., et al., Detectingmorphologically distinct oligomeric forms of alpha-synuclein. J BiolChem, 2009. 284(17): p. 11048-5). Single colonies were collected,individually grown and stored at −80° C.

Atomic force microscope (AFM) imaging—AFM imaging and analysis wereperformed as described previously (Wang, M. S., et al., Characterizingantibody specificity to different protein morphologies by AFM. Langmuir,2009. 25(2): p. 912-8). Aliquots were deposited and incubated for 10 minon freshly cleaved mica at room temperature before the mica surface waswashed extensively with DI water and dried with compressed nitrogenflow. To image phage binding specificity for the different tau isoforms,an additional stringent wash with 0.1% tween/DI water was performed toremove non-specific binding phage. The coated mica samples were imagedin air using a MultiMode AFM Nanoscope IIIA system (Veeco/Digitalinstruments, Santa Barbara, Calif.) operating in tapping mode usingsilicon AFM probes (VISTAprobes, nanoscience instruments).

Single clone screening with AFM—Following positive panning, a phagepreparation from each individual recovered clone was analyzed for targetbinding specificity by AFM. Aliquots of each phage were added to micacoated with either BSA, monomeric or trimeric tau. Samples showing thehighest binding levels toward trimeric tau, but no reactivity toward BSAor monomeric tau were selected for further characterization.

DNA sequence correction—DNA sequences of recovered clones were obtainedand compared with other known scFv sequences (Marks, J. D., et al.,By-passing immunization. Human antibodies from V-gene librariesdisplayed on phage. J Mol Biol, 1991. 222(3): p. 581-97). All recoveredclones from the positive panning step contained a missing base pair nearthe amino terminal of the scFv sequence resulting in a shift in thereading frame (FIG. 12). The reading frame shift was corrected inselected clones using polymerase chain reaction (PCR) with customizedprimers (FIG. 13). The forward primers encompass the NcoI site(5′-CCATGG-3′) upstream of scFv sequence and include the missing base,while the reverse primer encompasses the NotI site (5′-GCGGCCGC-3′)downstream of scFv sequence. The corrected scFv gene sequences wereligated into the pGEMT plasmid vector for sequencing to confirm thedesired DNA sequence, and then ligated into the pIT2 plasmid vectorwhich contains a hexahistidine tag and c-myc tag for protein expression.The pIT2 plasmids were transformed into either E. coli strain HB2151 forscFv expression or TG1 for phage expression.

Phage binding specificity assay—Binding specificities of the sequencecorrected phage clones were verified by AFM. Purified phage weredeposited and incubated on mica coated with the different tau speciesand imaged to confirm binding specificity as described above.

Soluble scFv production and purification—Production and purification ofthe sequence corrected scFv proteins were performed as describedpreviously (Barkhordarian, H., et al., Isolating recombinant antibodiesagainst specific protein morphologies using atomic force microscopy andphage display technologies. Protein Eng Des Sel, 2006. 19(11): p.497-502). Concentrated supernatant, periplasm and cell lysate fractionswere prepared separately and tested for presence of scFv. Most of thescFv was located in the periplasmic fraction, with lower amountsexcreted to the supernatant as expected (Kipriyanov, S. M., G.Moldenhauer, and M. Little, High level production of soluble singlechain antibodies in small-scale Escherichia coli cultures. J ImmunolMethods, 1997. 200(1-2): p. 69-77). All fractions containing scFv werepooled and purified using a Ni-NTA agarose beads column (Qiagen, 5 mLbeads for 1 L expression culture) and imidazole elution essentially asdescribed (Kasturirangan, S., S. Boddapati, and M. R. Sierks, Engineeredproteolytic nanobodies reduce Abeta burden and ameliorate Abeta-inducedcytotoxicity. Biochemistry, 2010. 49(21): p. 4501-8).

Dot blot assay with human brain tissue—Postmortem human brain samplesfrom the middle temporal gyms (MTG) of Alzheimer's disease (AD) andcognitively normal non-demented (ND) cases were generously provided byDr. Thomas Beach (Director of Banner/Sun Health Research Institute BrainBank). Brain extracts from the MTG of age-matched ND and AD patientswere homogenized in Tris-HCl/EDTA buffer with protease inhibitor. Thehomogenate was spun down to remove solids and the supernatant containingall the soluble protein was collected and adjusted to a total proteinconcentration of 3 mg/mL. Aliquots of 2 μL 3 mg/mL brain tissue weredotted on gridded nitrocellulose membrane and probed withanti-oligomeric tau scFv essentially as described (Zameer, A., et al.,Anti-oligomeric Abeta single-chain variable domain antibody blocksAbeta-induced toxicity against human neuroblastoma cells. J Mol Biol,2008. 384(4): p. 917-28). Samples were analyzed in triplicates usingpurified scFv. Reactivity of scFv with brain tissue samples was analyzedusing ImageJ and recorded in the form of densitometric value(Kasturirangan, S., et al., Isolation and characterization of antibodyfragments selective for specific protein morphologies from nanogramantigen samples. Biotechnol Prog, 2013. 29(2): p. 463-71). Each valuewas calibrated on a scale of 0 to 1 in which 0 denotes the backgroundand 1 denotes the positive control of anti-phosphorylas b (plb) scFv.

Mouse brain tissue—Brains from 5, 8, 11 months old wild-type mice and 5,9 and 13 months old 3×transgenic Alzheimer's (3×TG-AD) miceoverexpressing human tau P301L (Oddo, S., et al., Amyloid depositionprecedes tangle formation in a triple transgenic model of Alzheimer'sdisease. Neurobiol Aging, 2003. 24(8): p. 1063-70) were generouslyprovided by Dr. Travis Dunckley (Translational Genomics, Phoenix,Ariz.). Mouse hippocampus was homogenized as described above for humanbrain samples.

Phage biotinylation—Phage were biotinylated for enhanced signaldetection in ELISA using the EZ-Link Pentylamine-biotin kit (ThermoScientific). A 10¹¹ pfu/mL aliquot of phage stock (0.729 mg/ml) wasincubated with Pentylamine-Biotin (4.86mM final concentration) and1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) of0.1M final concentration at room temperature for 2 hours with stirring.Excess Pentylamine-Biotin and EDC were removed with desalting columns.

Capture ELISA—High affinity polystyrene microtiter 96-well plates werecoated with 100 of 0.3 mg/ml purified single clone scFv (captureantibody) and incubated at 37° C. for two hours. After the unbound scFvswere removed, the plates were washed three times with 0.1% tween/PBS.The plates were then blocked with 2% non-fat milk/PBS at 37° C. for onehour. After a tween/PBS wash, an aliquot of 100 μl/well of 0.2 mg/mlmouse brain homogenate (target analytes) was added, incubated at 37° C.for two hours and then washed with tween/PBS. PBS was used as a negativecontrol. A 100 μL/well aliquot of 10⁷ pfu/ml biotinylated phage(detection antibody) was incubated for coating at 37° C. for two hours.The wells were washed with tween/PBS, and then a 100 μL/well aliquot of0.5 μg/ml avidin-HRP was added and incubated at 37° C. for one hour. Theplates were washed again with tween/PBS and binding monitored using achemiluminescent ELISA kit (SuperSignal ELISA Femto Maximum SensitivitySubstrate (Thermo Scientific)). The chemiluminescent signal was read 1minute after addition to the mixture. The immunoreactivity signals werenormalized by dividing the absolute chemiluminescent readings of thesamples by that of PBS control. Within each independent experiment, themean signal obtained from all the wild-type mice samples was used as abaseline to normalize the transgenic mice signals.

To verify that the isolated scFvs were binding oligomeric tau in themouse brain tissue samples we used a sandwich ELISA where the scFvs wereused as a capture antibody and a commercially available monoclonalantibody against hyperphosphorylated tau aggregates, AT8 (ThermoScientific) was used as a detection antibody. AT8 binds tau forms withphosphorylated Ser202/Thr205 found in PHF-tau. An anti-mouse antibodyconjugated with horse radish peroxide(HRP) (Thermo Scientific) was usedto detect bound AT8.

Size analysis of individual phage target—To determine the size of thetarget antigen bound by individual phage particles, a 10 μI aliquot ofthe fibrillar tau aggregate mixture (19.5 μg/mL, 10×dilution of theoriginal prepared stock) was deposited on mica, and a 10 μl aliquot of10¹² pfu/ml phage was added, incubated and rinsed as described above.The aggregated mixture of rhTau 2N4R contained monomeric, oligomeric andfibrillar aggregates. AFM images (5 μm²) were obtained and processedusing Nanoscope Analysis. The diameter of each target antigen particlebound at the tip of the phage was calculated by taking the differencebetween the maximum height of the particle and the adjusted baseline. Atleast 6 different antigen particles for each individual clone weremeasured and averaged to determine the particle height of the targetantigen.

Statistical analysis—Samples were analyzed by one-way ANOVA with p<0.05standard and LSD post hoc significant differences test. All analyseswere performed with SPSS 21.0 (IBM Corp., Armonk, N.Y.)

Results and Discussion

Isolation of scFvs Selectively Binding Oligomeric Tau

We utilized an AFM based panning protocol that incorporates sequentialsubtractive and positive panning steps (FIG. 6A) to isolate scFvs thatselectively bind a toxic trimeric tau species. We first eliminated fromthe scFv library pool all those phage containing scFvs reactive with thecontrol protein (BSA) (FIG. 6B) and then monomeric tau. After thesubtractive panning steps, we isolated phage that selectively boundtrimeric rhTau 1N4R tau using a single positive panning step. Werecovered 96 phage clones from the positive panning step againsttrimeric tau (FIG. 6C). Phage from each of the 96 clones were preparedseparately and used to verify binding specificity for trimeric tau,monomeric tau and BSA by AFM. We selected twenty clones that selectivelyrecognized trimeric tau for further study. The DNA sequence of thetwenty clones were obtained to verify the presence of full length scFv,and six distinct full-length scFvs were selected for further analysis(H2, F9, D11, G12, H7, D4 and G12). Although all six clones containedcomplete scFv sequences, they all each lacked a DNA base pair shortlydownstream of the N-terminal NcoI site and the methionine start codon(FIG. 12) resulting in a reading frame shift. The pelB leader sequencecontains multiple methionine start codons in different reading framesthat may facilitate the expression of full length scFvs despite thealtered reading frame resulting from the missing base pair at theN-terminal. To enhance soluble scFvs expression efficiency, we correctedthe reading frame shift using PCR. We then verified that the eachsequence corrected scFv maintained the same binding specificity of theoriginal clone by AFM. Three sequence corrected clones F9T, D11C and H2Awere then selected for further studies based on binding specificity anddistinctive CDR sequences.

Verification of Binding Specificity to Tau Trimer

To verify that the F9T, D11C and H2A scFvs were selectively bindingtrimeric tau, we incubated a phage displayed version of each scFv with asample of aggregated tau and used AFM to determine the average height ofthe particles bound by each phage particle. We then compared the heightof the bound particles to the known height values of different tauaggregate species. The average height of at least 6 different boundantigens for each scFv was determined and compared to the size of knownoligomeric tau aggregates. The target antigen size for all three scFvscorrespond to the size of a rhTau 2N4R trimer (from 2.5 nm to 3.0 nm)providing further evidence that the scFvs selectively target trimerictau (FIG. 7).

Characterization of Binding Epitopes

Purified soluble scFv protein for each corrected scFv sequence had theexpected 29 kDa, the full length size for an scFv. Since the scFvs wereisolated against a synthetic oligomeric tau variant, we then testedwhether the purified F9T, D11C and H2A scFvs could recognize naturallyoccurring tau aggregates in brain tissue of an AD mouse model andwhether they cross-react with random proteins in brain. All three scFvclones bound oligomeric tau aggregates preferentially present inhippocampus tissue homogenates from 9-month old 3×TG AD mouse modelcompared to wild-type mice (FIG. 8). The bound aggregates were detectedusing the anti-tau antibody AT8 to verify that the scFvs wereselectively binding tau aggregates. Detection with AT8 also indicatesthat the tau aggregates in the brain tissue samples targeted by F9T,D11C and H2A can also be phosphorylated even though the initial antigentargets were not phosphorylated.

Since the three selected clones (i.e., F9T, D1 1C and H2A) containdistinctive CDR sequences, we determined whether they bind similar ordifferent epitopes on trimeric tau using a capture ELISA protocol wherepurified scFv was used as a capture antibody and the phage displayedversion of each scFv was used as a detection antibody. We testeddifferent combinations of capture and detection scFvs using the 3×TG-ADmouse brain homogenates as antigen. When F9T-displayed phage was used asthe detection antibody, strong signals were obtained when all threescFvs used used as capture antibodies. In contrast, when D11C-displayedphage was used as a detection antibody, lower signals were obtained withD11C and H2A as capture antibodies, and no signal with F9T; and whenH2A-displayed phage was used as a detection antibody, a lower signal wasobtained with D11C as the capture antibody and no signal with either F9Tor H2A as capture antibodies (FIG. 9). Since F9T-displayed phage gives astrong signal even when F9T scFv is used as the capture antibody, F9Trecognizes a trimer specific epitope that occurs in multiple locationson the tau aggregate. The antigen recognized by D11C may also havemultiple epitopes since D11C phage showed reactivity to tau aggregatescaptured by D11C scFv. However the antigen recognized by H2A may haveonly a single epitope since no signal was obtained with tau aggregatescaptured by the H2A scFv. Since F9T phage produced the strongestimmunoreactivity with brain extracts retained by all three scFvs ascapture antibody, we used F9T phage as the detection antibody in allfurther capture ELISAs.

Time Dependent Presence of Oligomeric Tau in AD Mouse Brain Tissue

We analyzed how oligomeric tau concentration varied with time in the3×TG-AD mice using the three scFvs against oligomeric tau. In the 3×TGmouse model, insoluble tau tangles are typically detected around 12-15months of age, however we find that oligomeric tau levels are alreadyhigh at 5-months of age, peak at 9-months and decline by 13-months (FIG.10). As expected we see similar trends with all three scFvs since eachscFv recognizes different epitopes of the same oligomeric tau species.Samples from age-matched wild-type mice did not show the presence of anyoligomeric tau reactive with these scFvs. The results from this mousemodel indicate that the concentration of oligomeric tau speciesincreases at early time points (5-9 months) before insoluble tau tanglesbegin to form, and then decreases after neurofibrillary tangles begin toform (12-15 months) suggesting that the oligomeric tau species may beincorporated into the NFTs. Since oligomeric tau aggregates are alreadypresent at 5-months well before presence of NFTs they have promise as anearly diagnostic for AD.

Analysis of Post-Mortem Human Brain Tissue

Since the scFvs effectively detected oligomeric tau species present inbrain tissue from an AD mouse model, we next probed post-mortem humanmiddle temporal gyms (MTG) extracts from different Braak stages for thepresence of oligomeric tau using the F9T scFv (FIG. 11). Oligomeric tauwas readily detected in the human brain samples using F9T. Interestinglythe concentration of oligomeric tau in the sampes increases withincreasing Braak stage as there is only minimal oligomeric tau in the NDBraak stage I-II samples, higher values in the ND Braak stage I-IIsamples with slight plaques, higher values again in AD samples withmoderate plaques (Braak stage III-IV) and the highest signals in ADsamples with heavy plaques (Braak stage V-VI). There is a significantdifference between the levels of oligomeric tau in the AD samplescompared to both the ND samples without plaques and the ND samples withslight plaques. These results indicate that morphology specific reagentssuch as F9T can be used to detect the presence of oligomeric tau speciesin human samples and have promise not only as early diagnostics for ADbut also to help stage progression of the disease.

Summary

Aggregates of Aβ and tau are the primary protein constituents of thehallmark senile plaques and neurofibrillary tangles of AD. While manystudies have focused on accumulation and aggregation of Aβ as aninitiating factor in AD pathogenesis and neuronal death with taudysfunction considered to be a downstream event following Aβaggregation, other studies suggest that tau interacts with Aβ toaccelerate the progression of AD, and that reducing aggregated taulevels are also important to ameliorate AD symptoms. Aβ and tauaggregation may be linked by separate mechanisms driven by a commonupstream cause. Numerous studies have implicated the role of solubleoligomeric Aβ species in mediating toxicity in AD, and evidence nowsuggests that oligomeric tau may also play toxic roles in AD. Recentstudies indicate that soluble tau species including oligomeric,prefibrillar and immature prefilament forms play more crucial roles inAD than the hallmark NFTs which instead may rather play an adaptive andprotective role. Oligomeric tau has been shown to have prion-likeself-propagating features and can be endocytosed into neurons where theycan induce endogenous tau pathology in vivo. Therefore the roles ofoligomeric and fibrillar tau species in AD progression is gettingincreasing attention and oligomeric tau is a promising therapeutictarget for AD. Because of the diversity of tau species that may bepresent in the human brain due to the alternative post-transcriptionalsplicing and post-translational modifications that may occur, there is acritical need to develop reagents that can selectively identifyindividual tau aggregate variants to probe the roles of the variousforms in disease progression and to assess their value as diagnostic andtherapeutic targets.

We previously reported that a trimeric, but not monomeric or dimeric tauspecies was neurotoxic at low nanomolar levels. Here we isolated threedifferent scFvs (F9T, D11C and H2A) that selectively recognize thistoxic trimeric tau species. All three scFvs have unique CDR sequences,bind to different epitopes on the tau aggregate and detect oligomerictau species in an AD mouse model by 5-months of age. While NFTs are ahallmark feature of AD, oligomeric tau species may play an intermediaterole in tau aggregation and have been shown to play an important role inneuronal toxicity, so identification and quantification of oligomerictau variants has great promise as an biomarker for early diagnosis ofAD. Here we show that the oligomeric tau levels can be used todifferentiate post-mortem human AD brain tissue samples from age-matchedcognitively normal cases. Quantification of oligomeric tau alsodistinguishes the post-mortem human samples according to Braak stagewhich is based on Aβ plaque and abnormal tau immunohistochemicalstaining.

Example 5

Trimeric Tau is Toxic to Human Neuronal Cells at Low NanomolarConcentrations

Alzheimer's disease (AD) is the most common form of dementia,characterized by progressive cognitive impairment, cerebral atrophy, andneuronal loss, with death generally occurring four to eight years afterdiagnosis. Two pathological hallmarks of AD, extracellular neuriticplaques primarily composed of amyloid beta (Aβ) and intracellularneurofibrillary tangles (NFTs) primarily composed of tau protein, wereoriginally identified in 1907 by Dr. Alzheimer. While great strides havebeen made in understanding the mechanisms that promote aggregation of Aβand tau into the hallmark plaques and tangles, comparatively littleprogress has been achieved in halting or curing the disease. Analysis offamilial AD cases implicated production of Aβ as a primary factor inprogression of AD, leading to the rise of the amyloid cascade hypothesiswhich states that Aβ misfolding and aggregation initiates ADpathogenesis and triggers other effects such as tau phosphorylation,aggregation, and tangle formation. The amyloid hypothesis had dominatedthe field for more than a decade and has driven numerous clinicalstudies for therapeutic interventions including several immunizationstudies targeting Aβ. However failure of several clinical trialstargeting Aβ has cast doubt on its relevance as a therapeutic target.Increasing evidence indicates that tau also plays an important role inthe progression of AD. Tau misfolding and aggregation can take placeindependently of amyloid formation, and in many cases the presence oftau lesions is associated with AD without presence of Aβ aggregates.Clearance of Aβ plaques without reducing soluble tau levels isinsufficient to ameliorate cognitive decline in double transgenic miceoverexpressing Aβ and tau P301L. These results among many othersindicate that oligomeric tau may be an important therapeutic target forAD.

Tau in its monomeric form is a microtubule associated protein crucialfor microtubule assembly and stabilization. Six major tau isoforms canbe generated by alternative posttranscriptional splicing of exon 2 andexon 3 on the N-terminal projection domain and of exon 10 (Repeat 2) onthe assembly domain (FIG. 14). Tau contains three or four similarrepeats in the microtubule binding domain (MBD) that binds to and helpspromote microtubule stability and function. For example, Repeat 2 andRepeat 3 contain hexapeptide motifs of PHF6* and PHF6, respectively(FIG. 14). These motifs increase the tendency to form β-sheet structuresthat can interact with tubulins to form microtubules and also facilitateself-assembly to generate oligomeric and higher-order aggregates. Tauisoforms with or without the second microtubule-binding repeat canaggregate, but only the isoforms with the second repeat can formextended oligomeric forms mediated by disulfide linkages due to theadditional cysteines in the second repeat (FIGS. 14 and 15). Therefore,in this study we utilized tau isoforms containing the second repeat unitto study the role of tau aggregation in neurotoxicity.

Hyperphosphorylation of tau is required for the release of tau frommicrotubules and its mislocalization to the somatodendritic compartmentenabling tau to self-associate into oligomers and higher-orderaggregates. However, the hyperphosphorylation of tau is not directlyrelated to its toxicity but rather a mechanism to regulate itsinteraction with tubulin to stabilize microtubules and to regulatetransport along microtubules. Expression of exogenous tau in maturehippocampal neurons leads to blockage of transport along microtubulesand degeneration of synapses that can be rescued by phosphorylation oftau by kinase MARK2 to unblock the microtubule tracks. Significantly,tau in the extracellular space is reported to be less phosphorylatedthan intracellular tau and more toxic in its dephosphorylated state.Extracellular oligomers of recombinant full-length human tau proteinwere shown to be neurotoxic in mice and impair memory consolidation, andsimilar work at other labs has shown similar effects with recombinanttau oligomers and tau oligomers composed of hyperphosphorylated tau fromAD brain. Thus, the hyperphosphorylation of tau associated with diseasemay be a causal factor in tau self-association into oligomers, but thehyperphosphorylation of tau in and of itself may not be the basis forthe toxicity of extracellular tau oligomers.

Neurofibrillary tangles (NFTs) have traditionally been correlated withneuronal loss and considered to be key intracellular indicators of AD.Approaches for targeting tau aggregation have focused on inhibitinghyperphosphorylation and fibril formation, reducing total tau levels, orstabilizing microtubules. However, accumulating evidence suggests thatsoluble oligomeric rather than insoluble fibrillar tau species areneurotoxic and play an important role in the onset and progression ofAD. Although NFTs are a hallmark feature of AD, they can exist in ADneurons for up to 20 to 30 years before postmortem confirmation andtherefore are less likely to induce immediate toxicity in AD brain. Inanimal models of tauopathy, the presence of NFTs does not correlate wellwith neuronal loss and memory deficits. Reduction in neuronal loss andimprovement in memory performance are observed despite an increase inNFTs. In addition, the presence of NFT pathology does not localize wellwith areas of neuronal loss, synapse loss or dysfunction in thehippocampus along with microglial activation occurs well before thepresence of NFTs. In contrast, oligomeric tau was implicated in numerousstudies as playing a key role in AD progression and to be a primaryinitiator of neurotoxicity and neurodegeneration. Oligomeric tau hasbeen identified in early stages of neuronal cytopathology in AD andclosely correlates with hyperphosphorylation on microtubule-bindingsites. Tau oligomers can propagate endogenous tau pathology throughoutthe brain similarly to prions, demonstrating their neuronal toxicity.The presence and concentrations of two tau oligomers (140 kDa and 170kDa) correlate with memory loss in various age rTg4510 mice. Oligomerictau also induces synaptic and mitochondrial dysfunction. Although tau ispredominantly intracellular, the role of extracellular tau is gainingattention as extracellular oligomeric tau can have acute effects onlong-term potentiation in hippocampal slices and can transmit pathologyto healthy neurons. Detection of oligomeric tau levels in human CSF andblood is also a promising AD diagnostic biomarkers along with total andhyperphosphorylated tau levels. Because of the important role ofoligomeric tau in AD and the recognition of the importance ofextracellular tau in disease, it is critical to identify the key toxictau species in disease etiology. Here we show our studies of theextracellular neurotoxicity of monomeric, dimeric, and trimeric forms oftwo four-repeat recombinant human tau variants to help identify the keytau species involved in the onset and progression of AD.

2. Materials and Methods

2.1. Recombinant Human Tau (rhTau) Preparation and Purification. rhTauwas purified as monomers from bacterial (BL21 DE3) clones with tauconstructs in the pET21B and pET29a vectors. Standard methods were usedto grow and induce the protein with 1 mM IPTG. Pelleted cells were lysedwith CelLytic B lysis buffer, lysozyme, benzonase, and proteaseinhibitors according to the manufacturer's protocol (Sigma Aldrich, St.Louis, Mo.). Cation exchange (GEHealthcare Life Sciences) was used forthe first step of purification with SPSepharose resin for both tauconstructs, and 300 mM NaCl in 25 mM Tris-HCl pH 7.4 was used to elutetau protein. Amicon Ultra Centrifugal Devices (Millipore) were used tobuffer-exchange the protein preparations into 50 mM Tris-HCl pH 7.4.Protein concentration was determined using a BCA assay (Thermo FisherScientific). Tau oligomers were generated by incubating tau monomers ata concentration of 5 μM in 50mM Tris buffer pH 7.4 with 100 mM NaCl at37° C. overnight. The monomeric and oligomeric species were resolved by6% PAGE, eluted, and buffer-exchanged into 50 mM Tris-HCl. Fractionswere analyzed by nonreducing SDS-PAGE to minimize degradation ofoligomeric proteins and silver staining to enhance the signal and toverify the purity of tau variants. Protein concentration was determinedusing the BCA assay.

2.2. Height Distribution Analysis. AFM sample preparation and imagingwere performed as described previously (H. Barkhordarian, S. Emadi, P.Schulz, and M. R. Sierks, “Isolating recombinant antibodies againstspecific proteinmorphologies using atomic force microscopy and phagedisplay technologies,” Protein Engineering, Design and Selection, vol.19, no. 11, pp. 497-502, 2006; A. Zameer, P. Schulz, M. S. Wang, and M.R. Sierks, “Single chain Fv antibodies against the 25-35A/β fragmentinhibit aggregation and toxicity of A/β42,” Biochemistry, vol. 45, no.38, pp. 11532-11539, 2006; S. Emadi, H. Barkhordarian, M. S. Wang, P.Schulz, and M. R. Sierks, “Isolation of a human single chain antibodyfragment against oligomeric α-synuclein that inhibits aggregation andprevents α-synuclein-induced toxicity,” Journal of Molecular Biology,vol. 368, no. 4, pp. 1132-1144, 2007; A. Zameer, S. Kasturirangan, S.Emadi, S. V. Nimmagadda, and M. R. Sierks, “Anti-oligomeric Aβsingle-chain variable domain antibody blocks Aβ-induced toxicity againsthuman neuroblastoma cells,” Journal of Molecular Biology, vol. 384, no.4, pp. 917-928, 2008; S. Emadi, S. Kasturirangan, M. S. Wang, P. Schulz,and M. R. Sierks, “Detecting morphologically distinct oligomeric formsof α-synuclein,”The Journal of Biological Chemistry, vol. 284, no. 17,pp. 11048-11058, 2009; M. S. Wang, A. Zameer, S. Emadi, and M. R.Sierks, “Characterizing antibody specificity to different proteinmorphologies by AFM,” Langmuir, vol. 25, no. 2, pp. 912-918, 2009.)Aliquots of 10 μL 0.5004 purified tau variants in 50 mM Tris-HCl bufferwere deposited on separate mica pieces for imaging using MultiMode AFMNanoscope IIIA system (Veeco/Digital instruments, Santa Barbara, Calif.)which was set in tapping mode and equipped with silicon AFM probes(VISTA probes, Nanoscience Instruments). Height distribution analysis ofthe different tau samples was fit to a normal distribution probabilitymodel using Gwyddion 2.20. All detectable protein molecules were assumedto be spherical and the height values approximate their diameters.

2.3. Cell Culture and Treatments. SH-SYSY human neuroblastoma cell lines(American Tissue Culture Collection) were cultivated in tissue cultureflask (Falcon by Becton Dickinson Labware). Cells were grown in a mediumcontaining 44% v/v Ham's F-12 (Irvine Scientific), 44% v/v MEM Earle'ssalts (Irvine Scientific), 10% v/v denatured fetal bovine serum (FBS)(Sigma Aldrich), 1% v/v MEM nonessential amino acids (Invitrogen), and1% v/v antibiotic/antimycotic (Invitrogen). Media were renewed onceevery two to three days. The cells were passaged to a new flask whenthey were confluent in the flask. For toxicity studies, the SH-SY5Ycells were seeded in a 48-well cell culture cluster plate (Costar byCorning Incorporated) with 5×104 cells/well in 300 μL fresh medium. Eachexperiment was conducted in triplicate. Cell density was estimated byreading a fixed volume on a hemocytometer. After growth in a 37° C.incubator for 24 hours, the tissue culture media were replaced withfresh serum-free media for the neurotoxicity test on nondifferentiatedcells. To investigate tau toxicity on cholinergic neurons, a duplicateset of the cultured cells was induced into cholinergic-like phenotype byincubation with retinoic acid at a final concentration of 10 μM for 3 to5 days (S. Emadi, S. Kasturirangan, M. S. Wang, P. Schulz, and M. R.Sierks, “Detecting morphologically distinct oligomeric forms ofα-synuclein,”The Journal of Biological Chemistry, vol. 284, no. 17, pp.11048-11058, 2009; S. Pahlman, J. C. Hoehner, E. Nanberg et al.,“Differentiation and survival influences of growth factors in humanneuroblastoma,” European Journal of Cancer A, vol. 31, no. 4, pp.453-458, 1995; M. Encinas, M. Iglesias, Y. Liu et al., “Sequentialtreatment of SH-SY5Y cells with retinoic acid and brain-derivedneurotrophic factor gives rise to fully differentiated, neurotrophicfactor-dependent, human neuron-like cells,” Journal of Neurochemistry,vol. 75, no. 3, pp. 991-1003, 2000;] S. P. Presgraves, T. Ahmed, S.Borwege, and J. N. Joyce, “Terminally differentiated SH-SY5Y cellsprovide a model system for studying neuroprotective effects of dopamineagonists,” Neurotoxicity Research, vol. 5, no. 8, pp. 579-598, 2003.).The cultivated nondifferentiated and cholinergic-like neurons weretreated with monomeric, dimeric, and trimeric variants of 1N4R and 2N4Rat final concentrations of 2.26 nM, 4.50 nM, 11.15 nM, and 15.50 nM. APBS negative control was used as a standard for subsequent LDH assayanalysis. Cultures were incubated with tau species at 37° C. and sampledat 3, 18, 24, and 48 hour time points by harvesting 30 μL/well aliquots5 of culture supernatant.

2.4. LDH Assay. The LDH protocol is adapted from a commercial kit (SigmaAldrich) based on the generic protocol of Decker and Lohmann-Matthes.The LDH assay was performed as described previously (A. Zameer, P.Schulz, M. S. Wang, and M. R. Sierks, “Single chain Fv antibodiesagainst the 25-35Aβ fragment inhibit aggregation and toxicity of Aβ42,”Biochemistry, vol. 45, no. 38, pp. 11532-11539, 2006.). Absorbance wasmeasured at 490 nm (reference wavelength 690 nm). Relative absorbancevalues were calculated by subtracting the reference values from thevalues obtained at 490 nm. LDII % values greater than 150 are consideredtoxic.

2.5. Statistical Analysis. The relative absorbance values of all sampleswere normalized to those of controls which were set as 100% for eachindependent experiment. Group mean values were analyzed by one-way ANOVAwith P <0.05 standard and LSD post hoc significant differences test. Allanalyses were performed with SPSS 21.0 (IBM Corp., Armonk, N.Y.).

3. Results

3.1. rhTau Aggregate Analysis. We expressed recombinant human tau in abacterial host system to eliminate any posttranslational phosphorylationof tau and therefore remove any potential effects that phosphorylationmay have on tau aggregation or loss of function. The resultingnonphosphorylated human recombinant tau (NPrhTau) monomers containreactive cysteine groups with free thiols, facilitating the formation ofintramolecular disulfide bonds to make stable nonreactive monomers andthe formation of intermolecular disulfide bonds to produce tau oligomersand higher degree aggregates (FIG. 15). The polymerization reaction iscontrolled by incubation time and protein concentration. The nonreactivemonomeric, dimeric, and trimeric forms of both the 2N4R and 1N4R splicevariants generate stable aggregate morphologies with defined sizeprofiles dependent on the degree of oligomerization and length of thesplice variant as evidenced by SDS-PAGE and AFM height distributionanalysis (FIG. 16). The oligomer heights increment for each additionalmonomeric tau unit is fixed within a certain isoform, which is 0.5nm for1N4R variants and 1.0 nm for the 2N4R variants (FIG. 16). The size ofeach respective 2N4R species is also larger than the corresponding 1N4Rspecies (FIG. 16) as expected given that tau 2N4R contains the extraN-terminal insert compared with the 1N4R variants.

3.2. Extracellular rhTau Induced Neurotoxicity Test. While neither themonomeric or dimeric forms of tau from either the 1N or 2N splicevariants displayed detectable toxicity, the trimeric form of bothvariants exerted marked toxicity toward nondifferentiated (FIG. 17(a))and retinoic acid induced cholinergic-like neurons (FIG. 17(b)) with LDHvalues well above the toxic threshold of 150 at low nanomolarconcentrations (11.15 nM, and 15.50 nM).The fulllength 2N4R trimeric tauform displayed significantly higher toxicity than the 1N4R trimeric formtoward nondifferentiated neurons (FIG. 17(a)), although the effect isdiminished in the cholinergic-like neurons (FIG. 17(b)). When trimerictau was added to nondifferentiated SH-SY5Y cells, an increase intoxicity was observed with time at the highest concentrations for boththe 1N4R (FIGS. 18(a)), and 2N4R (FIG. 18(b)) trimeric variants.However, when trimeric tau was added to the cholinergic-like neurons,the toxicity of the 1N (FIGS. 18(c)) and 2N (FIG. 18(d)) variants wasrelatively consistent over the first 24 hours, but increased after 48hours. Both variants of trimeric tau showed increased toxicity towardthe cholinergic-like neurons compared to the nondifferentiated neuronsat short incubation times (FIG. 19(a)) but the reverse was observed atlonger incubation times (FIG. 19(b)).

4. Discussion

While the amyloid cascade hypothesis has dominated studies into theetiology of AD over the last decade or more, the importance of tau inthe onset and progression of AD is steadily becoming more apparent. Taupathology has been observed in the absence of Aβ deposits in childrenand young adult cases, and tau aggregates in the entorhinal-hippocampalregions precede the onset of Aβ pathology. Numerous studies have shownthat various oligomeric forms of Aβ are toxic to neurons and can impaircognitive performance, thus implicating their potential role as valuablebiomarkers for diagnosing AD. Similar to the important role of varioussoluble oligomeric Aβ species in AD, different soluble oligomeric formsof tau may also play a critical role in AD, also causing neuronal lossand cognitive dysfunction. Therefore to facilitate diagnoses andtherapeutic treatments for AD, it is important to identify the key tauspecies involved in the onset and progression of the disease. Given thattau has multiple splice variants and posttranslational modificationsites, we attempted to simplify the complex diversity of tau forms byfocusing on two nonphosphorylated human recombinant tau isoforms, 1N4Rand 2N4R. These two four-repeat (4R) isoforms of tau both have all fourrepeats of the microtubule-associated domains and are more prone to formthe aggregates readily phosphorylated by brain protein kinases thanthose with only three repeats (3R) due to the presence of Repeat 2 witha microtubule-affinity enhancing hexapeptide motifs and an additionalcysteine that forms disulfide linkages to stabilize the aggregates.

The most disease-relevant tau material to use to study toxicity ofextracellular tau forms would be well characterized tau oligomerspurified from AD cerebrospinal fluid (CSF) using methods to preservetheir posttranslational modifications, including phosphorylation,glycation, ubiquitination, aggregation, and truncation. Preparationsfrom several non-AD and AD cases would be necessary to understand thesignificance of the results. Here we performed an initial study focusedspecifically on unmodified tau protein oligomers and control monomer tospecifically understand the relevance of oligomer structure toextracellular toxicity.

We determined the toxicity of the different tau variants using bothnondifferentiated and cholinergic-like neuroblastoma cell lines todetermine how aggregate size and cell phenotype affected toxicity.Cholinergic cells are particularly vulnerable in AD with significantneuronal loss in the nucleus basalis of Meynert (NBM), that is, thehippocampus and the cortex. NBM is enriched in cholinergic cells andundergoes degeneration and a significant decrease of acetylcholineproduction in AD. Decreased levels of acetylcholine and a number ofother cortical cholinergic markers lead to clinical dementia andimpairment in cognitive function, indicating that cholinergic cells areparticularly vulnerable in AD. Here we show that trimeric, but notmonomeric or dimeric, tau is toxic to neuronal cells at low nanomolarconcentrations and that the full-length 2N tau variant is more toxicthan the shorter 1Nvariant to nondifferentiated neurons (FIG. 17). Bothtrimeric tau variants cause toxicity to both nondifferentiated SH-SY5Ycells and retinoic acid induced cholinergic-like neurons when tau wasapplied extracellularly at nanomolar levels (FIG. 18). However, thecultured cholinergic-like neurons show increased susceptibility totrimeric tau induced toxicity at short incubation times compared withsimilar nondifferentiated neurons (FIG. 19(a)), perhaps partiallyaccounting for the increased vulnerability of cholinergic-like neuronsin AD. Since the nondifferentiated cells were equally susceptible totrimeric tau induced toxicity at longer incubation times (FIG. 19(b)),these results suggest that toxicity of extracellular trimeric tau is notdependent on receptors or proteins specifically associated withcholinergic cells but that toxicity might be facilitated by them. Ourresults are consistent with a recent study showing that low molecularweight (LMW) misfolded tau species exclusive of monomeric tau can beendocytosed by neurons and transported both anterogradely andretrogradely to induce endogenous tau pathology in vivo while fibrillartau and brain-derived filamentous tau cannot be endocytosed. Thissuggests that tau toxicity may be spread through cells in certain brainregions by endocytosis of trimeric and larger oligomeric forms of tauand that this uptake is facilitated in cholinergic neurons. Neuronaltoxicity of oligomeric tau may share similar properties to that ofoligomeric Aβ where the critical feature involved in neuronal toxicityis the aggregation state of the protein more than posttranslationalmodifications.

While there are a wide variety of tau variants that occur in vivoincluding different posttranslational modifications, splice variants,and aggregated species, this study begins to more systematically probethe role of selected tau variants in AD. Further studies are needed todetermine the contribution of splice variants and AD-specificposttranslational modifications found in extracellular tau to thetoxicity of the tau variants and to how these tau variants affect otherneuronal models including primary neurons or induced pluripotent stemcells. Well characterized reagents that can selectively identifyspecific tau variants and morphologies will be useful for these furtherstudies.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. An antibody or antibody fragment that specifically recognizesoligomeric tau but does not bind monomeric tau, fibrillar tau ornon-disease associated forms of tau.
 2. The antibody or antibodyfragment of claim 1, wherein the oligomeric tau is dimeric tau ortrimeric tau.
 3. The antibody or antibody fragment of claim 2, whereinthe oligomeric tau is trimeric tau.
 4. The antibody or antibody fragmentof claim 1, wherein the oligomeric tau is soluble.
 5. The antibody orantibody fragment of claim 1, wherein said antibody fragment is isolatedaccording to a method comprising the steps of: a. a negative panning ofa scFV phage library wherein said negative panning eliminates phage thatbind to non-desired antigens wherein said negative panning comprisesserially contacting phage with: (i) a generic protein; and (ii)mononeric forms of tau; and monitoring the binding of said phage to thegeneric protein and monomeric forms of tau using Atomic Force Microscope(AFM) Imaging and repeating steps (i) and (ii) until no phage isobserved binding to antigen by said AFM imaging to produce an aliquot ofphage; b. contacting the aliquot of phage with tau oligomers andincubating for time sufficient to allow binding of phage to saidoligomers; and c. eluting the bound phage particles from step (b).
 6. Anantibody or antibody fragment isolated according to a method comprisingthe steps of: (a) negative panning a scFV phage library comprisingserially contacting phage with: (i) a generic protein; and (ii)mononeric forms of tau; and until less than 5% of the phage is observedbinding to antigen, which produces an aliquot of phage; (b) positivepanning of the aliquot from step (a) comprising contacting the aliquotof phage from step (a) with tau oligomers, and incubating for timesufficient to allow binding of phage to said brain derived tauoligomers; and (c) eluting the bound phage particles from step (b). 7.The antibody or antibody fragment of claim 5, wherein the tau oligomeris trimeric tau 4N1R.
 8. The antibody or antibody fragment of claim 5,wherein the generic protein is bovine serum albumin (BSA).
 9. Theantibody or antibody fragment of claim 5, wherein the negative panningfurther comprises serially contacting phage with brain derived controlsamples that do not contain oligomeric tau.
 10. The antibody or antibodyfragment of claim 6, wherein the observing of the binding of the phageto the antigen is by using Atomic Force Microscope (AFM) Imaging. 11.The antibody or antibody fragment of claim 6, wherein the negativepanning is repeated until less than 0-10% phage was observed by AFMimaging as binding to antigen in step (a).
 12. The antibody fragment ofclaim 1, wherein said antibody fragment does not contain the constantdomain region of an antibody.
 13. An antibody fragment comprising aminoacid sequence SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, or SEQ ID NO:19.
 14. The antibody fragment ofclaim 13, wherein the antibody fragment comprises amino acid sequenceSEQ ID NO:1 , SEQ ID NO:9 or SEQ ID NO:11.
 15. A binding molecule thatbinds to oligomeric tau and does not bind monomelic tau, fibrillar tauor non-disease associated forms of tau, wherein the binding moleculecomprises the sequence of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, or SEQ ID NO:19.
 16. A method ofinhibiting the aggregation of tau comprising contacting a compositionthat comprises tau oligomers with an antibody, antibody fragment orbinding molecule of claim
 1. 17. The method of claim 16, wherein saidaggregation of tau is in a cell.
 18. The method of claim 16, whereinsaid aggregation of tau is in brain tissue.
 19. The method of claim 16,wherein said contacting with an antibody, antibody fragment or bindingmolecule decreases the rate of formation of tau aggregates as comparedto said rate in the absence of composition or binding molecule.
 20. Amethod of detecting the presence of tau in a physiological samplecomprising contacting a sample with an antibody, antibody fragment or abinding molecule of claim 1 and determining the binding of saidcomposition with said tissue sample wherein binding of said compositionto said tissue sample is indicative of the presence of tau oligomers insaid tissue sample wherein said presence of said tau oligomers isindicative of early stage AD, frontotemporal dementia, other tauopathiesor neurodegeneration following traumatic brain injury.
 21. The method ofclaim 20, wherein the physiological sample is brain tissue, serum,cerebrospinal fluid (CSF), blood, urine or saliva.
 22. The method ofclaim 16, wherein the tau oligomers are dimeric trimeric tau.
 23. Themethod of claim 22, wherein the tau oligomers are trimeric tau.
 24. Themethod of claim 22, wherein the tau oligomers are soluble.
 25. A methodof preventing or inhibiting the accumulation of tau in the brain of amammal comprising administering to said mammal a composition comprisingan antibody fragment or a binding molecule of claim
 1. 26. The method ofclaim 20, wherein the physiological sample is spinal tissue.